CA2973674A1 - Production of steviol glycosides in recombinant hosts - Google Patents

Production of steviol glycosides in recombinant hosts Download PDF

Info

Publication number
CA2973674A1
CA2973674A1 CA2973674A CA2973674A CA2973674A1 CA 2973674 A1 CA2973674 A1 CA 2973674A1 CA 2973674 A CA2973674 A CA 2973674A CA 2973674 A CA2973674 A CA 2973674A CA 2973674 A1 CA2973674 A1 CA 2973674A1
Authority
CA
Canada
Prior art keywords
seq
polypeptide
compound
steviol
ent
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
CA2973674A
Other languages
French (fr)
Inventor
Kim OLSSON
Ernesto SIMON
Michael DALGAARD MIKKELSEN
Simon CARLSEN
Veronique Douchin
Swee Chuang LIM
Louis During
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Evolva Holding SA
Original Assignee
Evolva AG
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Evolva AG filed Critical Evolva AG
Publication of CA2973674A1 publication Critical patent/CA2973674A1/en
Pending legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/44—Preparation of O-glycosides, e.g. glucosides
    • C12P19/56—Preparation of O-glycosides, e.g. glucosides having an oxygen atom of the saccharide radical directly bound to a condensed ring system having three or more carbocyclic rings, e.g. daunomycin, adriamycin
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H15/00—Compounds containing hydrocarbon or substituted hydrocarbon radicals directly attached to hetero atoms of saccharide radicals
    • C07H15/20—Carbocyclic rings
    • C07H15/24—Condensed ring systems having three or more rings
    • C07H15/256—Polyterpene radicals
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1048—Glycosyltransferases (2.4)
    • C12N9/1051—Hexosyltransferases (2.4.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P15/00—Preparation of compounds containing at least three condensed carbocyclic rings
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y204/00—Glycosyltransferases (2.4)
    • C12Y204/01—Hexosyltransferases (2.4.1)

Abstract

The invention relates to recombinant microorganisms and methods for producing steviol glycosides, glycosylated ent-kaurenol, and glycosylated ent-kaurenoic acid.

Description

PRODUCTION OF STEVIOL GLYCOSIDES IN RECOMBINANT HOSTS
BACKGROUND OF THE INVENTION
Field of the Invention [0001] This disclosure relates to recombinant production of steviol glycosides and steviol glycoside precursors in recombinant hosts. In particular, this disclosure relates to production of steviol glycosides comprising steviol-13-0-glucoside (13-SMG), stevio1-19-0-glucoside (19-SMG), steviol-1,2-bioside, steviol-1,3-bioside, 1,2-stevioside, 1,3-stevioside, rubusoside (Rubu), rebaudioside A (RebA), rebaudioside B (RebB), rebaudioside D (RebD), rebaudioside E (RebE), rebaudioside M (RebM), rebaudioside Q (RebQ), rebaudioside 1 (Rebl), di-glycosylated steviol, tri-glycosylated steviol, tetra-glycosylated steviol, penta-glycosylated steviol, hexa-glycosylated steviol, hepta-glycosylated steviol, glycosylated ent-kaurenol, glycosylated ent-kaurenoic acid, and/or isomers thereof in recombinant hosts.
Description of Related Art
[0002] Sweeteners are well known as ingredients used most commonly in the food, beverage, or confectionary industries. The sweetener can either be incorporated into a final food product during production or for stand-alone use, when appropriately diluted, as a tabletop sweetener or an at-home replacement for sugars in baking. Sweeteners include natural sweeteners such as sucrose, high fructose corn syrup, molasses, maple syrup, and honey and artificial sweeteners such as aspartame, saccharine, and sucralose. Stevia extract is a natural sweetener that can be isolated and extracted from a perennial shrub, Stevie rebaudiana. Stevia is commonly grown in South America and Asia for commercial production of stevia extract.
Stevia extract, purified to various degrees, is used commercially as a high intensity sweetener in foods and in blends or alone as a tabletop sweetener.
[0003] Chemical structures for several steviol glycosides are shown in Figure 1, including the diterpene steviol and various steviol glycosides. Extracts of the Stevia plant generally comprise steviol glycosides that contribute to the sweet flavor, although the amount of each steviol glycoside often varies, inter alia, among different production batches.
[0004] As recovery and purification of steviol glycosides from the Stevia plant have proven to be labor intensive and inefficient, there remains a need for a recombinant production system that can accumulate high yields of desired steviol glycosides, such as RebD
and RebM. There also remains a need for improved production of steviol glycosides in recombinant hosts for commercial uses. As well, there remains a need for identifying enzymes selective towards particular substrates to produce one or more specific steviol glycosides. In some aspects, there remains a need to increase the catalytic capability of enzymes with 19-0 glycosylation activity in order to produce higher yields of steviol glycosides.
SUMMARY OF THE INVENTION
[0005] It is against the above background that the present invention provides certain advantages and advancements over the prior art.
[0006] Although this invention as disclosed herein is not limited to specific advantages or functionalities, the invention provides a recombinant host cell, comprising at least one recombinant gene that is:
(a) a gene encoding a UGT91D2e polypeptide having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:11;
(b) a gene encoding a chimeric polypeptide having at least 70% sequence identity to the amino acid sequence set forth in SEQ ID NO:17 or SEQ ID NO:18;
(c) a gene encoding a UGT85C2 polypeptide having at least 55% sequence identity to the amino acid sequence set forth in SEQ ID NO:7; and/or (d) a gene encoding a UGT76G1 polypeptide having at least 50% sequence identity to the amino acid sequence set forth in SEQ ID NO:9;
wherein the recombinant host cell is capable of producing a steviol glycoside, glycosylated ent-kaurenol compound, and/or a glycosylated ent-kaurenoic acid compound in a cell culture broth.
[0007] In one aspect of the recombinant host cell disclosed herein, the UGT91D2e polypeptide comprises a UGT91D2e polypeptide having at least one amino acid substitution at residues 93, 99, 114, 144, 148, 152, 195, 196, 199, 211, 213, 221, 286, 384, 426, 438, or 466 of SEQ ID NO:11.
[0008] In one aspect of the recombinant host cell disclosed herein, the polypeptide comprises a UGT85C2 polypeptide having at least one amino acid substitution at residues 21, 48, 49, 84, 86, 87, 91, 92, 95, 122, 334, or 334 of SEQ ID NO:7.
[0009] In one aspect of the recombinant host cell disclosed herein, the polypeptide comprises a UGT76G1 polypeptide having at least one amino acid substitution at residues 23, 26, 55, 146, 257, 283, and 337 of SEQ ID NO:9.
[0010] In one aspect of the recombinant host cell disclosed herein, the UGT91D2e polypeptide comprises one or more of the UGT91D2e polypeptide variants comprising: P93V, S99I, 5114F, T144K, T144L, T144M, A148K, M152T, L195G, L195C, L1955, L195N, L195V, V196P, K199C, L211H, L211M, L211I, L211C, L211T, L213E, S221I, V286C, V286N, V2865, G384W, G384K, G384Y, E426G, E438H, 3438M or A466V of SEQ ID NO:11.
[0011] In one aspect of the recombinant host cell disclosed herein, the polypeptide comprises one or more of the UGT85C2 polypeptide variants comprising: Q21L, Q21T, Q21V, F485, F48H, F48Y, F48R, F48Q, F48W, F48T, I49V, 584G, 584A, 584T, S84C, 584P, 584N, 584V, P86R, P86G, I87H, I87P, I87M, I87Y, L91K, L91R, L91T, L92F, L92I, L92M, I95K, F1225, L3345 or L334M of SEQ ID NO:7.
[0012] In one aspect of the recombinant host cell disclosed herein, the polypeptide comprises one or more of the UGT76G1 polypeptide variants comprising: Q23H, I26W, T146G, H155L, L257G, S253W, T284G, 5283N, K337P or T55K of SEQ ID NO:9.
[0013] In one aspect the recombinant host cell disclosed herein further comprises at least one recombinant gene that is:
(a) a gene encoding a geranylgeranyl diphosphate synthase (GGPPS) polypeptide;
(b) a gene encoding an ent-copalyl diphosphate synthase (CDPS) polypeptide;
(c) a gene encoding an ent-kaurene synthase (KS) polypeptide;
(d) a gene encoding an ent-kaurene oxidase (KO) polypeptide;
(e) a gene encoding a cytochrome P450 reductase (CPR) polypeptide; and (f) a gene encoding an ent-kaurenoic acid hydroxylase (KAH) polypeptide;
(g) a gene encoding a UGT74G1 polypeptide; and/or (h) a gene encoding an EUGT11 polypeptide;
wherein the recombinant host cell capable of producing a steviol glycoside, glycosylated ent-kaurenol compound, and/or a glycosylated ent-kaurenoic acid compound in a cell culture broth.
[0014] In one aspect of the recombinant host cell disclosed herein, (a) the GGPPS polypeptide comprises a polypeptide having at least 70%
identity to an amino acid sequence set forth in SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID
NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, or SEQ ID NO:116;
(b) the CDPS polypeptide comprises a polypeptide having at least 70%
identity to an amino acid sequence set forth in SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ
ID
NO:40, or SEQ ID NO:42;
(c) the KS polypeptide comprises a polypeptide having at least 70% identity to an amino acid sequence set forth in SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ
ID
NO:50, or SEQ ID NO:52;
(d) the KO polypeptide comprises a polypeptide having at least 70% identity to an amino acid sequence set forth in SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:117, SEQ ID
NO:66, SEQ ID NO:68, SEQ ID NO:70, SEQ ID NO:72, SEQ ID NO:74, or SEQ ID
NO:76;
(e) the CPR polypeptide comprises a polypeptide having at least 70%
identity to an amino acid sequence set forth in SEQ ID NO:78, SEQ ID NO:80, SEQ ID NO:82, SEQ
ID
NO:84, SEQ ID NO:86, SEQ ID NO:88, SEQ ID NO:90, SEQ ID NO:92;
(f) the KAH polypeptide comprises a polypeptide having at least 70%
identity to an amino acid sequence set forth in SEQ ID NO:94, SEQ ID NO:97, SEQ ID NO:100, SEQ ID
NO:101, SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO:104, SEQ ID NO:106, SEQ ID
NO:108, SEQ ID NO:110, SEQ ID NO:112, or SEQ ID NO:114;
(g) the UGT74G1 polypeptide comprises a polypeptide having at least 55%
identity to an amino acid sequence set forth in SEQ ID NO:4;
(h) the EUGT11 polypeptide comprises a polypeptide having at least 65%
identity to an amino acid sequence set forth in SEQ ID NO:16.
[0015] In one aspect of the recombinant host cell disclosed herein, the cell culture broth comprises:
(a) the steviol glycoside, glycosylated ent-kaurenol compound, and/or the glycosylated ent-kaurenoic acid compound produced by the recombinant host cell, (b) glucose, fructose and/or sucrose; and/or (c) supplemental nutrients comprising trace metals, vitamins, salts, yeast nitrogen base (YNB), and/or amino acids.
[0016] In one aspect of the recombinant host cell disclosed herein, the recombinant host comprises a plant cell, a mammalian cell, an insect cell, a fungal cell, an algal cell, or a bacterial cell.
[0017] In one aspect of the recombinant host cell disclosed herein, the bacterial cell comprises Escherichia cells, Lactobacillus cells, Lactococcus cells, Comebacterium cells, Acetobacter cells, Acinetobacter cells, or Pseudomonas cells.
[0018] In one aspect of the recombinant host cell disclosed herein, the fungal cell comprises a yeast cell.
[0019] In one aspect of the recombinant host cell disclosed herein, the yeast cell is a cell from Saccharomyces cerevisiae, Schizosaccharomyces pombe, Yarrowia lipolytica, Candida glabrata, Ashbya gossypii, Cyberlindnera jadinii, Pichia pastoris, Kluyveromyces lactis, Hansenula polymorpha, Candida boidinii, Arxula adeninivorans, Xanthophyllomyces dendrorhous, or Candida albicans species.
[0020] In one aspect of the recombinant host cell disclosed herein, the yeast cell is a Saccharomycete.
[0021] In one aspect of the recombinant host cell disclosed herein, the yeast cell is a cell from the Saccharomyces cerevisiae species.
[0022] The invention also provides a method of producing a steviol glycoside, glycosylated ent-kaurenol compound, and/or glycosylated ent-kaurenoic acid compound in a cell culture broth, comprising growing the recombinant host cell disclosed herein in a culture medium, under conditions in which one or more of the genes are expressed;
wherein at least one of the genes is a recombinant gene;
wherein the steviol glycoside, glycosylated ent-kaurenol compound, and/or the glycosylated ent-kaurenoic acid compound is produced by the recombinant host cell.
[0023] In one aspect of the methods disclosed herein, one or more of the genes is constitutively expressed and/or expression of one or more of the genes is induced.
[0024] The invention also provides a method for producing a steviol glycoside, glycosylated ent-kaurenol compound, and/or the glycosylated ent-kaurenoic acid compound comprising whole-cell bioconversion of plant-derived components or synthetic steviol or steviol glycosides using one or more of:

(a) a UGT91D2e polypeptide having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:11;
(b) a chimeric polypeptide having at least 70% sequence identity to the amino acid sequence set forth in SEQ ID NO:17 or SEQ ID NO:18;
(c) a UGT85C2 polypeptide having at least 55% sequence identity to the amino acid sequence set forth in SEQ ID NO:7; and/or (d) a UGT76G1 polypeptide having at least 50% sequence identity to the amino acid sequence set forth in SEQ ID NO:9;
wherein at least one of the polypeptides is a recombinant polypeptide.
[0025] In one aspect of the methods disclosed herein, the whole cell is the recombinant host cell disclosed herein.
[0026] In one aspect of the methods disclosed herein, the recombinant host cell is grown in a fermentor at a temperature for a period of time, wherein the temperature and period of time facilitate the production of the steviol glycoside, glycosylated ent-kaurenol compound, and/or glycosylated ent-kaurenoic acid compound.
[0027] The invention also provides an in vitro method for producing a steviol glycoside, glycosylated ent-kaurenol compound, and/or the glycosylated ent-kaurenoic acid compound, comprising adding one or more of:
(a) a UGT91D2e polypeptide having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:11;
(b) a chimeric polypeptide having at least 70% sequence identity to the amino acid sequence set forth in SEQ ID NO:17 or SEQ ID NO:18;
(c) a UGT85C2 polypeptide having at least 55% sequence identity to the amino acid sequence set forth in SEQ ID NO:7; and/or (d) a UGT76G1 polypeptide having at least 50% sequence identity to the amino acid sequence set forth in SEQ ID NO:9, and plant-derived components or synthetic steviol or steviol glycosides to a reaction mixture;
wherein at least one of the polypeptides is a recombinant polypeptide; and (b) synthesizing steviol glycoside, glycosylated ent-kaurenol compound, and/or the glycosylated ent-kaurenoic acid compound in the reaction mixture.
[0028] In one aspect, methods disclosed herein further comprise isolating the steviol glycoside, glycosylated ent-kaurenol compound, and/or the glycosylated ent-kaurenoic acid compound, alone or in combination from the cell culture broth.
[0029] In one aspect of the methods disclosed herein, the isolating step comprises:
(a) providing the cell culture broth comprising the steviol glycoside, glycosylated ent-kaurenol compound, and/or the glycosylated ent-kaurenoic acid compound alone or in combination;
(b) separating a liquid phase of the cell culture broth from a solid phase of the cell culture broth to obtain a supernatant comprising the steviol glycoside, glycosylated ent-kaurenol compound, and/or the glycosylated ent-kaurenoic acid compound alone or in combination;
(c) providing one or more adsorbent resins, comprising providing the adsorbent resins in a packed column; and (d) contacting the supernatant of step (b) with the one or more adsorbent resins in order to obtain at least a portion of the steviol glycoside, glycosylated ent-kaurenol compound, and/or the glycosylated ent-kaurenoic acid compound alone or in combination thereby isolating the steviol glycoside, glycosylated ent-kaurenol compound, and/or the glycosylated ent-kaurenoic acid compound alone or in combination.
[0030] In one aspect, methods disclosed herein further comprise recovering the the steviol glycoside, glycosylated ent-kaurenol compound, and/or the glycosylated ent-kaurenoic acid compound alone or a composition comprising the steviol glycoside, glycosylated ent-kaurenol compound, and/or the glycosylated ent-kaurenoic acid compound.
[0031] In one aspect of the methods disclosed herein, the recovered composition is enriched for the steviol glycoside, glycosylated ent-kaurenol compound, and/or the glycosylated ent-kaurenoic acid compound relative to a steviol glycoside composition of Stevie plant and has a reduced level of non-steviol glycoside Stevie plant-derived components relative to a plant-derived stevia extract.
[0032] In one aspect of the methods disclosed herein, the cell culture broth comprises:

(a) one or more steviol glycosides, glycosylated ent-kaurenol compounds, and/or glycosylated ent-kaurenoic acid compounds produced by the recombinant host cell disclosed herein, (b) glucose, fructose, and/or sucrose; and/or (c) supplemental nutrients comprising trace metals, vitamins, salts, YNB, and/or amino acids.
[0033] In one aspect of the methods disclosed herein, the reaction mixture comprising:
(a) one or more steviol glycosides, glycosylated ent-kaurenol compounds, and/or a glycosylated ent-kaurenoic acid compounds produced in the reaction mixture;
(b) a UGT polypeptide;
(c) UDP-glucose, UDP-rhamnose, UDP-xylose, and/or N-acetyl-glucosamine;
and/or (d) reaction buffer and/or salts.
[0034] In one aspect of the methods disclosed herein, the recombinant host cell comprises a plant cell, a mammalian cell, an insect cell, a fungal cell, an algal cell, or a bacterial cell.
[0035] In one aspect of the methods disclosed herein, the bacterial cell comprises Escherichia cells, Lactobacillus cells, Lactococcus cells, Comebacterium cells, Acetobacter cells, Acinetobacter cells, or Pseudomonas cells.
[0036] In one aspect of the methods disclosed herein, the fungal cell comprises a yeast cell.
[0037] In one aspect of the methods disclosed herein, the yeast cell is a cell from Saccharomyces cerevisiae, Schizosaccharomyces pombe, Yarrowia lipolytica, Candida glabrata, Ashbya gossypii, Cyberlindnera jadinii, Pichia pastoris, Kluyveromyces lactis, Hansenula polymorpha, Candida boidinii, Arxula adeninivorans, Xanthophyllomyces dendrorhous, or Candida albicans species.
[0038] In one aspect of the methods disclosed herein, the yeast cell is a Saccharomycete.
[0039] In one aspect of the methods disclosed herein, the yeast cell is a cell from the Saccharomyces cerevisiae species.
[0040] In one aspect of the recombinant hosts and methods disclosed herein, (a) the steviol glycoside comprises 13-SMG, 19-SMG, Stevio1-1,2-bioside, Stevio1-1,3-bioside, 1,2-stevioside, 1,3-stevioside, rubusoside, RebA, RebB, RebD, RebE, RebM, di-glycosylated tri-glycosylated steviol, tetra-glycosylated steviol, penta-glycosylated steviol, hexa-glycosylated steviol, hepta-glycosylated steviol, and/or isomers thereof;
(b) the glycosylated ent-kaurenol compound comprises di-glycosylated ent-kaurenol, tri-glycosylated ent-kaurenol, and/or isomers thereof; and/or (c) the glycosylated ent-kaurenoic acid compound comprises di-glycosylated ent-kaurenoic acid, tri-glycosylated ent-kaurenoic acid, and/or isomers thereof.
[0041] In one aspect of the recombinant hosts and methods disclosed herein, (a) the di-glycosylated steviol comprises compound 2.23 of Table 1;
(b) the tri-glycosylated steviol comprises compound 3.1 and/or compound 3.34 of Table 1;
(c) the tetra-glycosylated steviol comprises compound 4.26 and/or compound 4.33 of Table 1;
(d) the penta-glycosylated steviol comprises compound 5.22, compound 5.24, and/or compound 5.25 of Table 1;
(e) the hexa-glycosylated steviol comprises compound 6.1 and/or compound 6.23 of Table 1;
(f) the hepta-glycosylated steviol comprises compound 7.2, compound 7.5, and/or compound 7.13 of Table 1;
(9) the glycosylated ent-kaurenoic acid compound comprises compound KA3.1, compound KA3.2, and/or compound KA2.7 of Table 1; and/or (h) the glycosylated ent-kaurenol compound comprises compound KL2.8 and/or compound KL3.1 co-eluted with compound KL3.6 of Table 1.
[0042] In one aspect of the recombinant hosts and methods disclosed herein, (a) compound 4.26 has the structure:

HO
OH
G

if OH
MOHO
(b) compound 5.22 has the structure:

HO
OH
HO

HO
HO
OH
*10 Ho ,0 HO

HOI/OH
HO OH
(c) compound 6.1 has the structure:
HO

HO OH

HO

HO
HO
OH
HC
HO

H):' OH
HOHO

(d) compound 7.2 has the structure:
HO
HO
HO
HO

HO
HO
ecH31111t HO
HO
HO. Ho HOHO
(e) compound 7.5 has the structure:
HO
HO

OH

HO
HOIHO
HO
OH
iocH"
HO
HO

HOH0 .
OH
HOHO
(f) compound KA3.1 has the structure:

4' OH

'OH
OH
(g) compound KA3.2 has the structure:

H
... HO
õ==== OH

, OH ;and (h) compound KL3.1 has the structure:
H
H0b ,..
HO
0 p 0 ,,,OH
o../
OH

p_ 0H
OH
-OH

OH .
[0043] In one aspect of the recombinant hosts and methods disclosed herein, (a) the tri-glycosylated ent-kaurenoic acid comprises a compound having the structure:
H
.2 . H V JO H 0 H

'OH
p....
H OH
OH
(b) the penta-glycosylated steviol comprises a compound having the structure:

HO

HO

HO

HO
OH

HO
HO

'NH
HO OH
(c) the hexa-glycosylated steviol comprises a compound having the structure:
HO
HO
"H7I54".

HO

HO
HO
OH
VAIIV -OOP
HsC
HO
HOO

HO OH

; and (d) the hepta-glycosylated steviol comprises a compound having the structure:
HO
HO
H 0 HC:.--ft=\/' thC

H
HO"
ON
HON
[0044] The invention also provides a steviol glycoside composition produced by the recombinant host cell disclosed herein or the method disclosed herein, wherein the composition has a steviol glycoside composition enriched for RebD, RebM, or isomers thereof relative to a steviol glycoside composition of Stevie plant and has a reduced level of non-steviol glycoside Stevie plant-derived components relative to a plant-derived stevia extract.
[0045] The invention also provides a cell culture broth comprising:
(a) the recombinant host cell disclosed herein; and (b) one or more steviol glycosides, glycosylated ent-kaurenol compounds, and/or glycosylated ent-kaurenoic acid compounds produced by the recombinant host cell;
wherein one or more steviol glycosides is present at a concentration of at least 1 mg/liter of the culture broth.
[0046] The invention also provides a cell culture broth comprising:
(a) one or more steviol glycosides, glycosylated ent-kaurenol compounds, and/or glycosylated ent-kaurenoic acid compounds produced by the recombinant host cell disclosed herein, (b) glucose, fructose, sucrose, xylose, ethanol, and/or glycerol; and/or (c) supplemental nutrients comprising trace metals, vitamins, salts, YNB, and/or amino acids.
[0047] The invention also provides a cell lysate comprising:
(a) one or more steviol glycosides, glycosylated ent-kaurenol compounds, and/or glycosylated ent-kaurenoic acid compounds produced by the recombinant host cell disclosed herein, (b) glucose, fructose, sucrose, xylose, ethanol, glycerol, uridine diphosphate (UDP)-glucose, UDP-rhamnose, UDP-xylose, and/or N-acetyl-glucosamine; and/or (c) supplemental nutrients comprising trace metals, vitamins, salts, YNB, and/or amino acids.
[0048] The invention also provides a reaction mixture comprising:
(a) one or more steviol glycosides, glycosylated ent-kaurenol compounds, and/or a glycosylated ent-kaurenoic acid compounds produced in the reaction mixture;

(b) a UGT polypeptide;
(c) glucose, fructose, sucrose, xylose, ethanol, glycerol, uridine diphosphate (UDP)-glucose, UDP-rhamnose, UDP-xylose, and/or N-acetyl-glucosamine; and/or (d) reaction buffer and/or salts.
[0049] These and other features and advantages of the present invention will be more fully understood from the following detailed description taken together with the accompanying claims.
It is noted that the scope of the claims is defined by the recitations therein and not by the specific discussion of features and advantages set forth in the present description.
BRIEF DESCRIPTION OF THE DRAWINGS
[0050] The following detailed description of the embodiments of the present invention can be best understood when read in conjunction with the following drawings, where like structure is indicated with like reference numerals and in which:
[0051] Figure 1 shows a schematic of the engineered biosynthetic pathway for producing steviol in yeast from geranylgeranyl diphosphate using geranylgeranyl diphosphate synthase (GGPPS), ent-copalyl diphosphate synthase (CDPS), ent-kaurene synthase (KS), ent-kaurene oxidase (KO), and ent-kaurenoic acid hydroxylase (KAH) polypeptides.
[0052] Figure 2 shows representative steviol glycoside glycosylation reactions catalyzed by suitable uridine 5'-diphospho (UDP) glycosyl transferases (UGT) enzymes and chemical structures for several steviol glycoside compounds.
[0053] Figure 3 shows the steviol synthetic intermediate, ent-kaurenol, and its bioconversion product, ent-kaurenoic acid, for the steviol pathway step catalyzed by a KO, along with potential glycosylation by-products (mono-, di-, and/or tri-glycosylated ent-kaurenol and mono-, di-, or tri-glycosylated ent-kaurenoic acid).
[0054] Figure 4A shows accumulation of ent-kaurenoic acid+2GIc (#7), ent-kaurenoic acid+3GIc (isomer 1), and ent-kaurenoic acid+3GIc (isomer 2) by a steviol glycoside-producing S. cerevisiae strain deleted of UGT85C2 (SEQ ID NO:7). Figure 4B shows accumulation of 19-SMG by a steviol glycoside-producing S. cerevisiae strain deleted of UGT85C2 (SEQ ID NO:7).
Figure 40 shows accumulation of steviol, stevio1+2GIc (#23), and stevio1+3GIc (#34) by a steviol glycoside-producing S. cerevisiae strain deleted of UGT85C2 (SEQ ID NO:7). See Example 6.
[0055] Figure 5 shows conversion of steviol to rubusoside by bacterial lysates comprising UGT85C2 variants. Bacterial lysates were incubated with steviol for 24 h. See Example 7.
[0056] Figure 6A shows production of RebM, RebD, RebA, RebB, 13-SMG, and rubusoside in a steviol glycoside-producing strain expressing UGT76G1 H155L (gray bars), compared to the control steviol glycoside-producing strain expressing wild-type UGT76G1 (black bars).
Figure 6B shows production of 1,2-bioside, rubusoside (Rubu), RebG, and RebE
in a steviol glycoside-producing strain expressing UGT76G1 H155L (gray bars), compared to a control strain expressing wild-type UGT76G1 (black bars). Figure 60 shows production of quantifiable steviol glycosides (13-SMG + 1,2-bioside + Rubu + RebG + RebB + RebA + RebE +
RebD +
RebM) and RebD plus RebM titers in a steviol glycoside-producing strain expressing UGT76G1 H155L (gray bars), compared to a control strain expressing wild-type UGT76G1 (black bars).
Figure 6D shows production of a tri-glycosylated steviol molecule (stevio1+3G1c (#1)), a tetra-glycosylated steviol molecule (stevio1+4G1c (#26)), three penta-glycosylated steviol molecules (stevio1+5G1c (#22), stevio1+5G1c (#24), and stevio1+5G1c (#25)), two hexa-glycosylated steviol molecules (stevio1+6G1c (isomer 1) and stevio1+6G1c (#23)), and two hepta-glycosylated steviol molecules (stevio1+7G1c (isomer 2) and stevio1+7G1c (#13)) in a steviol glycoside-producing strain expressing UGT76G1 H155L (gray bars), compared to a control strain expressing wild-type UGT76G1 (black bars). See Example 9.
[0057] Figure 7A shows NMR-elucidated structures of tri-glycosylated ent-kaurenoic acid (Ent-Kaurenoic Acid+3G1c (isomers 1 and 2)), ent-kaurenoic acid+2G1c+1G1cNAc, and tri-glycosylated ent-kaurenol (ent-kaureno1+3G1c (isomer 1)). Figure 7B shows NMR-elucidated structures of stevio1+6G1c (isomer 1) and stevio1+7G1c (isomer 2). Figure 70 shows NMR-elucidated structures of stevio1+6G1c (isomer 4) and stevio1+7G1c (isomer 5).
Figure 7D shows NMR-elucidated structures of stevio1+4G1c+1G1cNAc (#11) and stevio1+4G1c (#26). Figure 7E
shows NMR-elucidated structures of stevio1+5G1c (#22) and stevio1+7G1c (#14).
See Examples 6, 8, and 9.
[0058] Figures 8A, 8B, and 80 show a 11-1 NMR spectrum and 1H and 130 NMR
chemical shifts (in ppm) for ent-kaurenoic acid+3G1c (isomer 1). Figures 8D, 8E, and 8F
show a 1H NMR
spectrum and 1H and 130 NMR chemical shifts (in ppm) for ent-kaurenoic acid+3G1c (isomer 2).
Figures 8G, 8H, and 81 show a 1H NMR spectrum and 1H and 130 NMR chemical shifts (in ppm) for ent-kaurenoic acid+2G1c+1G1cNAc. Figures 8J, 8K, and 8L show a 1H NMR
spectrum and 1H
and 130 NMR chemical shifts (in ppm) for ent-kaureno1+3G1c (isomer 1). Figures 8M, 8N, 80, and 8P show a 1H NMR spectrum and 1H and 130 NMR chemical shifts (in ppm) for stevio1+6G1c (isomer 1). Figures 8Q, 8R, 8S, and 8T show a 1H NMR spectrum and 1H and 130 NMR
chemical shifts (in ppm) for stevio1+7GIc (isomer 2). Figures 8U, 8V, 8W, and 8X show a 1H
NMR spectrum and 1H and 130 NMR chemical shifts (in ppm) for stevio1+6GIc (isomer 4).
Figures 8Y, 8Z, 8AA, and 8AB show a 1H NMR spectrum and 1H and 130 NMR
chemical shifts (in ppm) for stevio1+7GIc (isomer 5). Figures 8AC, 8AD, 8AE, and 8AF show a 1H
NMR
spectrum and 1H and 130 NMR chemical shifts (in ppm) for stevio1+4G1c+1GIcNAc (#11).
Figures 8AG, 8AH, 8A1, and 8AJ show a 1H NMR spectrum and 1H and 130 NMR
chemical shifts (in ppm) for stevio1+4GIc (#26). Figures 8AK, 8AL, 8AM, and 8AN show a 1H NMR
spectrum and 1H and 130 NMR chemical shifts (in ppm) for stevio1+5GIc (#22). Figures 8A0, 8AP, 8AQ, and 8AR show a 1H NMR spectrum and 1H and 130 NMR chemical shifts (in ppm) for stevio1+7GIc (#14). See Examples 6, 8, and 9.
[0059]
Figure 9A shows accumulation of ent-kaurenoic acid+2GIc (#7), ent-kaurenoic acid+3GIc (isomer 1), and ent-kaurenoic acid+3GIc (isomer 2) in S. cerevisiae expressing UGT76G1 variants. Figure 9B shows accumulation of ent-kaureno1+2GIc (#8) and ent-kaureno1+3G1c (isomer 1) co-eluted with ent-kaureno1+3GIc (#6) in S.
cerevisiae expressing UGT76G1 variants. See Example 8.
[0060]
Figure 10A shows accumulation of 1,2-stevioside, RebG, stevio1+3GIc (#1), stevio1+4GIc (#26), stevio1+5GIc (#22), stevio1+5GIc (#24), stevio1+5GIc (#25), stevio1+6GIc (isomer 1), and stevio1+6GIc (#23) in S. cerevisiae expressing RebD-producing variants.
Figure 10B shows accumulation of 1,2-stevioside, RebG, stevio1+3GIc (#1), stevio1+4GIc (#26), stevio1+5GIc (#22), stevio1+5GIc (#24), stevio1+5GIc (#25), stevio1+6GIc (isomer 1), and stevio1+6GIc (#23) in S. cerevisiae expressing RebM-producing variants. Figure 100 shows accumulation of 13-SMG, 1,2-bioside, rubusoside, RebA, RebB, RebD, RebE, and RebM in S. cerevisiae expressing UGT76G1 variants. See Example 8.
[0061]
Figure 11A shows accumulation of ent-kaurenoic acid+2GIc (#7), ent-kaurenoic acid+3GIc (isomer 1), ent-kaurenoic acid+3GIc (isomer 2), ent-kaureno1+2GIc (#8), and ent-kaureno1+3G1c (isomer 1) co-eluted with ent-kaureno1+3GIc (#6) in an S.
cerevisiae steviol glycoside production strain (control strain comprised three copies of wild-type UGT76G1 (SEQ
ID NO:9); variant strains comprised two copies of wild-type UGT76G1 and one copy of a UGT76G1 variant). Figure 11B shows total levels of glycosylated ent-kaurenoic acid (ent-kaurenoic acid+2GIc (#7) + ent-kaurenoic acid+3GIc (isomer 1) + ent-kaurenoic acid+3GIc (isomer 2)) in an S. cerevisiae steviol glycoside production strain expressing UGT76G1 variants.
Figure 110 shows total levels of glycosylated ent-kaurenol (ent-kaureno1+3GIc (isomer 1) co-eluted with ent-kaureno1+3GIc (#6) and ent-kaureno1+2GIc (#8) in an S.
cerevisiae steviol glycoside production strain expressing UGT76G1 variants. Figure 11D shows accumulation of 1,2-bioside, 1,2-stevioside, stevio1+3GIc (#1), stevio1+4GIc (#26), stevio1+5GIc (#22), stevio1+5GIc (#24), stevio1+5GIc (#25), stevio1+6GIc (isomer 1), stevio1+6GIc (#23), stevio1+7GIc (isomer 2), and stevio1+7GIc (isomer 5) in an S. cerevisiae steviol glycoside production strain expressing UGT76G1 variants. Figure 11E shows accumulation of 13-SMG, 1,2-bioside, rubusoside, RebG, RebA, RebB, RebD, RebE, and RebM in an S. cerevisiae steviol glycoside production strain expressing UGT76G1 variants. See Example 8.
[0062] Skilled artisans will appreciate that elements in the Figures are illustrated for simplicity and clarity and have not necessarily been drawn to scale. For example, the dimensions of some of the elements in the Figures can be exaggerated relative to other elements to help improve understanding of the embodiment(s) of the present invention.
DETAILED DESCRIPTION OF THE INVENTION
[0063] Before describing the present invention in detail, a number of terms will be defined.
As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. For example, reference to a "nucleic acid" means one or more nucleic acids.
[0064] It is noted that terms like "preferably," "commonly," and "typically" are not utilized herein to limit the scope of the claimed invention or to imply that certain features are critical, essential, or even important to the structure or function of the claimed invention. Rather, these terms are merely intended to highlight alternative or additional features that can or cannot be utilized in a particular embodiment of the present invention.
[0065] For the purposes of describing and defining the present invention it is noted that the term "substantially" is utilized herein to represent the inherent degree of uncertainty that can be attributed to any quantitative comparison, value, measurement, or other representation. The term "substantially" is also utilized herein to represent the degree by which a quantitative representation can vary from a stated reference without resulting in a change in the basic function of the subject matter at issue.
[0066] Methods well known to those skilled in the art can be used to construct genetic expression constructs and recombinant cells according to this invention. These methods include in vitro recombinant DNA techniques, synthetic techniques, in vivo recombination techniques, and polymerase chain reaction (FOR) techniques. See, for example, techniques as described in Green & Sambrook, 2012, MOLECULAR CLONING: A LABORATORY MANUAL, Fourth Edition, Cold Spring Harbor Laboratory, New York; Ausubel et al., 1989, CURRENT
PROTOCOLS IN MOLECULAR BIOLOGY, Greene Publishing Associates and Wiley Interscience, New York, and PCR Protocols: A Guide to Methods and Applications (Innis et al., 1990, Academic Press, San Diego, CA).
[0067] As used herein, the terms "polynucleotide," "nucleotide,"
"oligonucleotide," and "nucleic acid" can be used interchangeably to refer to nucleic acid comprising DNA, RNA, derivatives thereof, or combinations thereof, in either single-stranded or double-stranded embodiments depending on context as understood by the skilled worker.
[0068] As used herein, the terms "microorganism," "microorganism host,"
"microorganism host cell," "recombinant host," and "recombinant host cell" can be used interchangeably. As used herein, the term "recombinant host" is intended to refer to a host, the genome of which has been augmented by at least one DNA sequence. Such DNA sequences include but are not limited to genes that are not naturally present, DNA sequences that are not normally transcribed into RNA or translated into a protein ("expressed"), and other genes or DNA
sequences which one desires to introduce into a host. It will be appreciated that typically the genome of a recombinant host described herein is augmented through stable introduction of one or more recombinant genes. Generally, introduced DNA is not originally resident in the host that is the recipient of the DNA, but it is within the scope of this disclosure to isolate a DNA segment from a given host, and to subsequently introduce one or more additional copies of that DNA into the same host, e.g., to enhance production of the product of a gene or alter the expression pattern of a gene. In some instances, the introduced DNA will modify or even replace an endogenous gene or DNA sequence by, e.g., homologous recombination or site-directed mutagenesis.
Suitable recombinant hosts include microorganisms.
[0069] As used herein, the term "recombinant gene" refers to a gene or DNA
sequence that is introduced into a recipient host, regardless of whether the same or a similar gene or DNA
sequence may already be present in such a host. "Introduced," or "augmented"
in this context, is known in the art to mean introduced or augmented by the hand of man. Thus, a recombinant gene can be a DNA sequence from another species or can be a DNA sequence that originated from or is present in the same species but has been incorporated into a host by recombinant methods to form a recombinant host. It will be appreciated that a recombinant gene that is introduced into a host can be identical to a DNA sequence that is normally present in the host being transformed, and is introduced to provide one or more additional copies of the DNA to thereby permit overexpression or modified expression of the gene product of that DNA. In some aspects, said recombinant genes are encoded by cDNA. In other embodiments, recombinant genes are synthetic and/or codon-optimized for expression in S. cerevisiae.
[0070] As used herein, the term "engineered biosynthetic pathway" refers to a biosynthetic pathway that occurs in a recombinant host, as described herein. In some aspects, one or more steps of the biosynthetic pathway do not naturally occur in an unmodified host. In some embodiments, a heterologous version of a gene is introduced into a host that comprises an endogenous version of the gene.
[0071] As used herein, the term "endogenous" gene refers to a gene that originates from and is produced or synthesized within a particular organism, tissue, or cell.
In some embodiments, the endogenous gene is a yeast gene. In some embodiments, the gene is endogenous to S. cerevisiae, including, but not limited to S. cerevisiae strain S288C. In some embodiments, an endogenous yeast gene is overexpressed. As used herein, the term "overexpress" is used to refer to the expression of a gene in an organism at levels higher than the level of gene expression in a wild type organism. See, e.g., Prelich, 2012, Genetics 190:841-54. In some embodiments, an endogenous yeast gene, for example ADH, is deleted.
See, e.g., Giaever & Nislow, 2014, Genetics 197(2):451-65. As used herein, the terms "deletion," "deleted," "knockout," and "knocked out" can be used interchangabley to refer to an endogenous gene that has been manipulated to no longer be expressed in an organism, including, but not limited to, S. cerevisiae.
[0072] As used herein, the terms "heterologous sequence" and "heterologous coding sequence" are used to describe a sequence derived from a species other than the recombinant host. In some embodiments, the recombinant host is an S. cerevisiae cell, and a heterologous sequence is derived from an organism other than S. cerevisiae. A heterologous coding sequence, for example, can be from a prokaryotic microorganism, a eukaryotic microorganism, a plant, an animal, an insect, or a fungus different than the recombinant host expressing the heterologous sequence. In some embodiments, a coding sequence is a sequence that is native to the host.
[0073] A "selectable marker" can be one of any number of genes that complement host cell auxotrophy, provide antibiotic resistance, or result in a color change.
Linearized DNA fragments of the gene replacement vector then are introduced into the cells using methods well known in the art (see below). Integration of the linear fragments into the genome and the disruption of the gene can be determined based on the selection marker and can be verified by, for example, FOR or Southern blot analysis. Subsequent to its use in selection, a selectable marker can be removed from the genome of the host cell by, e.g., Cre-LoxP systems (see, e.g., Gossen et al., 2002, Ann. Rev. Genetics 36:153-173 and U.S. 2006/0014264). Alternatively, a gene replacement vector can be constructed in such a way as to include a portion of the gene to be disrupted, where the portion is devoid of any endogenous gene promoter sequence and encodes none, or an inactive fragment of, the coding sequence of the gene.
[0074] As used herein, the terms "variant" and "mutant" are used to describe a protein sequence that has been modified at one or more amino acids, compared to the wild-type sequence of a particular protein.
[0075] As used herein, the term "inactive fragment" is a fragment of the gene that encodes a protein having, e.g., less than about 10% (e.g., less than about 9%, less than about 8%, less than about 7%, less than about 6%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, less than about 1%, or 0%) of the activity of the protein produced from the full-length coding sequence of the gene. Such a portion of a gene is inserted in a vector in such a way that no known promoter sequence is operably linked to the gene sequence, but that a stop codon and a transcription termination sequence are operably linked to the portion of the gene sequence. This vector can be subsequently linearized in the portion of the gene sequence and transformed into a cell. By way of single homologous recombination, this linearized vector is then integrated in the endogenous counterpart of the gene with inactivation thereof.
[0076] As used herein, the term "steviol glycoside" refers to rebaudioside A (RebA) (CAS #
58543-16-1), rebaudioside B (RebB) (CAS # 58543-17-2), rebaudioside C (RebC) (CAS #
63550-99-2), rebaudioside D (RebD) (CAS # 63279-13-0), rebaudioside E (RebE) (CAS #
63279-14-1), rebaudioside F (RebF) (CAS # 438045-89-7), rebaudioside M (RebM) (CAS #
1220616-44-3), rubusoside (CAS # 63849-39-4), dulcoside A (CAS # 64432-06-0), rebaudioside 1 (Rebl) (MassBank Record: FU000332), rebaudioside Q (RebQ), 1,2-stevioside (CAS #57817-89-7), 1,3-stevioside (RebG), 1,2-bioside (MassBank Record: FU000299), 1,3-bioside, stevio1-13-0-glucoside (13-SMG), steviol-19-0-glucoside (19-SMG), a di-glycosylated steviol, a tri-glycosylated steviol, a tetra-glycosylated steviol, a penta-glycosylated steviol, a hexa-glycosylated steviol, a hepta-glycosylated steviol, and/or isomers thereof.
See Figure 2; see also, Steviol Glycosides Chemical and Technical Assessment 69th JECFA, 2007, prepared by Harriet Wallin, Food Agric. Org. See Figure 2, Figure 7, Figure 8, and Table 1; see also, Steviol Glycosides Chemical and Technical Assessment 69th JECFA, 2007, prepared by Harriet Wallin, Food Agric. Org. Glycosylated steviol compounds can comprise one or more glucose, N-acetylglucosamine (GIcNAc), rhamnose, and/or xylose moieties. Non-limiting examples of steviol glycosides that can be produced by methods described herein are shown in Table 1, Figure 7, and Figure 8.
[0077] As used herein, the term "glycosylated ent-kaurenol compound" refers to di-glycosylated ent-kaurenol or tri-glycosylated ent-kaurenol. As used herein, the term "glycosylated ent-kaurenoic acid compound" refers to di-glycosylated ent-kaurenoic acid or tri-glycosylated ent-kaurenoic acid. See Figure 7, Figure 8, and Table 1.
Glycosylated ent-kaurenol compounds and glycosylated ent-kaurenoic acid compounds can comprise one or more glucose, GIcNAc, rhamnose, and/or xylose moieties. Non-limiting examples of glycosylated ent-kaurenol compounds and glycosylated ent-kaurenoic acid compounds that can be produced by methods described herein are shown in Table 1, Figure 7, and Figure 8.
[0078] As used herein, the terms "steviol glycoside precursor" and "steviol glycoside precursor compound" are used to refer to intermediate compounds in the steviol glycoside biosynthetic pathway. Steviol glycoside precursors include, but are not limited to, geranylgeranyl diphosphate (GGPP), ent-copalyl-diphosphate, ent-kaurene, ent-kaurenol, ent-kaurenal, ent-kaurenoic acid, and steviol. See Figure 1. In some embodiments, steviol glycoside precursors are themselves steviol glycoside compounds. For example, 19-SMG, rubusoside, stevioside, and RebE are steviol glycoside precursors of RebM. See Figure 2.
Steviol glycosides and/or steviol glycoside precursors can be produced in vivo (i.e., in a recombinant host), in vitro (i.e., enzymatically), or by whole cell bioconversion. As used herein, the terms "produce" and "accumulate" can be used interchangeably to describe synthesis of steviol glycosides and steviol glycoside precursors in vivo, in vitro, or by whole cell bioconversion.
[0079] As used herein, the term "cell culture broth" can be used to refer to a liquid that can support or has supported growth of a host cell, including, but not limited to, a yeast host cell.
The components of a cell culture broth can include, for example, a steviol glycoside, a glycosylated ent-kaurenol compound, and/or a glycosylated ent-kaurenoic acid compound produced by the host cell, glucose, fructose, sucrose, trace metals, vitamins, salts, yeast nitrogen base (YNB), and/or amino acids.
[0080] As used herein, the term "cell lysate" can be used to refer to a fluid comprising the components of a lysed cell, i.e., a cell whose membrane has been disrupted chemically or mechanically. A cell lysate can further comprise a steviol glycoside, a glycosylated ent-kaurenol compound, and/or a glycosylated ent-kaurenoic acid compound produced by the host cell, glucose, fructose, sucrose, xylose, rhamnose, uridine diphosphate (UDP)-glucose, UDP-rhamnose, UDP-xylose, GIcNAc, trace metals, vitamins, salts, YNB, and/or amino acids. In some aspects, a cell lysate is a yeast cell lysate, such as an S. cerevisiae cell lysate, or a bacterial cell lysate, such as an E. coli cell lysate.
[0081] As used herein, the term "reaction mixture" refers to a solution for conducting an in vitro reaction. The components of a reaction mixture can include, but are not limited to, a steviol glycoside, a glycosylated ent-kaurenol compound, a glycosylated ent-kaurenoic acid compound, a polypeptide such as a UGT polypeptide, UDP-glucose, UDP-rhamnose, UDP-xylose, GIcNAC, a buffer, and/or salts.
[0082] Recombinant steviol glycoside-producing Saccharomyces cerevisiae (S.
cerevisiae) strains are described in WO 2011/153378, WO 2013/022989, WO 2014/122227, and WO
2014/122328. Methods of producing steviol glycosides in recombinant hosts, by whole cell bio-conversion, and in vitro are also described in WO 2011/153378, WO 2013/022989, WO
2014/122227, and WO 2014/122328.
[0083] In some embodiments, steviol glycosides and/or steviol glycoside precursors are produced in vivo through expression of one or more enzymes involved in the steviol glycoside biosynthetic pathway in a recombinant host. For example, a steviol-producing recombinant host expressing one or more of a gene encoding a GGPPS polypeptide, a gene encoding a CDPS
polypeptide, a gene encoding a KS polypeptide, a gene encoding a KO
polypeptide, a gene encoding a KAH polypeptide, a gene encoding a CPR polypeptide, and a gene encoding a UGT
polypeptide can produce a steviol glycoside and/or steviol glycoside precursors in vivo. See, e.g., Figures 1 and 2. The skilled worker will appreciate that one or more of these genes can be endogenous to the host provided that at least one (and in some embodiments, all) of these genes is a recombinant gene introduced into the recombinant host.
[0084] A recombinant host described herein can comprise a gene encoding a polypeptide capable of synthesizing geranylgeranyl pyrophosphate (GGPP) from farnesyl diphosphate (FPP) and isopentenyl diphosphate (IPP), a gene encoding a polypeptide capable of synthesizing ent-copalyl dirophosphate from GGPP; a gene encoding a polypeptide capable of synthesizing ent-kaurene from ent-copalyl pyrophosphate, a gene encoding a polypeptide capable of synthesizing ent-kaurenoic acid from ent-kaurene, a gene encoding a polypeptide capable of synthesizing steviol from ent-kaurenoic acid; and/or a gene encoding a polypeptide capable of converting NADPH to NADP+. A GGPPS polypeptide can synthesize GGPP
from FPP and IPP. A CDPS polypeptide can synthesize ent-copalyl dirophosphate from GGPP. A
KS polypeptide can synthesize ent-kaurene from ent-copalyl pyrophosphate. A KO
polypeptide can synthesize ent-kaurenoic acid from ent-kaurene. A KAH polypeptide can synthesize steviol from ent-kaurenoic acid. A CPR polypeptide can convert NADPH to NADP+.
[0085] In another example, a recombinant host expressing a gene encoding a GGPPS
polypeptide, a gene encoding a CDPS polypeptide, a gene encoding a KS
polypeptide, a gene encoding a KO polypeptide, a gene encoding a KAH polypeptide, and a gene encoding a CPR
polypeptide can produce steviol in vivo. See, e.g., Figure 1. The skilled worker will appreciate that one or more of these genes can be endogenous to the host provided that at least one (and in some embodiments, all) of these genes is a recombinant gene introduced into the recombinant host.
[0086] In another example, a recombinant host expressing a gene encoding a GGPPS
polypeptide, a gene encoding a CDPS polypeptide, a gene encoding a KS
polypeptide, a gene encoding a KO polypeptide, a gene encoding a KAH polypeptide, a gene encoding a CPR
polypeptide, and one or more of a gene encoding a UGT polypeptide can produce a steviol glycoside in vivo. See, e.g., Figures 1 and 2. The skilled worker will appreciate that one or more of these genes can be endogenous to the host provided that at least one (and in some embodiments, all) of these genes is a recombinant gene introduced into the recombinant host.
[0087] In some aspects, the GGPPS polypeptide comprises a polypeptide having an amino acid sequence set forth in SEQ ID NO:20 (which can be encoded by the nucleotide sequence set forth in SEQ ID NO:19), SEQ ID NO:22 (which can be encoded by the nucleotide sequence set forth in SEQ ID NO:21), SEQ ID NO:24 (which can be encoded by the nucleotide sequence set forth in SEQ ID NO:23), SEQ ID NO:26 (which can be encoded by the nucleotide sequence set forth in SEQ ID NO:25), SEQ ID NO:28 (which can be encoded by the nucleotide sequence set forth in SEQ ID NO:27), SEQ ID NO:30 (which can be encoded by the nucleotide sequence set forth in SEQ ID NO:29), SEQ ID NO:32 (which can be encoded by the nucleotide sequence set forth in SEQ ID NO:31), or SEQ ID NO:116 (which can be encoded by the nucleotide sequence set forth in SEQ ID NO:115).
[0088] In some aspects, the CDPS polypeptide comprises a polypeptide having an amino acid sequence set forth in SEQ ID NO:34 (which can be encoded by the nucleotide sequence set forth in SEQ ID NO:33), SEQ ID NO:36 (which can be encoded by the nucleotide sequence set forth in SEQ ID NO:35), SEQ ID NO:38 (which can be encoded by the nucleotide sequence set forth in SEQ ID NO:37), SEQ ID NO:40 (which can be encoded by the nucleotide sequence set forth in SEQ ID NO:39), or SEQ ID NO:42 (which can be encoded by the nucleotide sequence set forth in SEQ ID NO:41). In some embodiments, the CDPS polypeptide lacks a chloroplast transit peptide.
[0089] In some aspects, the KS polypeptide comprises a polypeptide having an amino acid sequence set forth in SEQ ID NO:44 (which can be encoded by the nucleotide sequence set forth in SEQ ID NO:43), SEQ ID NO:46 (which can be encoded by the nucleotide sequence set forth in SEQ ID NO:45), SEQ ID NO:48 (which can be encoded by the nucleotide sequence set forth in SEQ ID NO:47), SEQ ID NO:50 (which can be encoded by the nucleotide sequence set forth in SEQ ID NO:49), or SEQ ID NO:52 (which can be encoded by the nucleotide sequence set forth in SEQ ID NO:51).
[0090] In some embodiments, a recombinant host comprises a gene encoding a CDPS-KS
polypeptide. In some aspects, the CDPS-KS polypeptide comprises a polypeptide having an amino acid sequence set forth in SEQ ID NO:54 (which can be encoded by the nucleotide sequence set forth in SEQ ID NO:53), SEQ ID NO:56 (which can be encoded by the nucleotide sequence set forth in SEQ ID NO:55), or SEQ ID NO:58 (which can be encoded by the nucleotide sequence set forth in SEQ ID NO:57).
[0091] In some aspects, the KO polypeptide comprises a polypeptide having an amino acid sequence set forth in SEQ ID NO:60 (which can be encoded by the nucleotide sequence set forth in SEQ ID NO:59), SEQ ID NO:62 (which can be encoded by the nucleotide sequence set forth in SEQ ID NO:61), SEQ ID NO:117 (which can be encoded by the nucleotide sequence set forth in SEQ ID NO:63 or SEQ ID NO:64), SEQ ID NO:66 (which can be encoded by the nucleotide sequence set forth in SEQ ID NO:65), SEQ ID NO:68 (which can be encoded by the nucleotide sequence set forth in SEQ ID NO:67), SEQ ID NO:70 (which can be encoded by the nucleotide sequence set forth in SEQ ID NO:69), SEQ ID NO:72 (which can be encoded by the nucleotide sequence set forth in SEQ ID NO:71), SEQ ID NO:74 (which can be encoded by the nucleotide sequence set forth in SEQ ID NO:73), or SEQ ID NO:76 (which can be encoded by the nucleotide sequence set forth in SEQ ID NO:75).
[0092] In some aspects, the CPR polypeptide comprises a polypeptide having an amino acid sequence set forth in SEQ ID NO:78 (which can be encoded by the nucleotide sequence set forth in SEQ ID NO:77), SEQ ID NO:80 (which can be encoded by the nucleotide sequence set forth in SEQ ID NO:79), SEQ ID NO:82 (which can be encoded by the nucleotide sequence set forth in SEQ ID NO:81), SEQ ID NO:84 (which can be encoded by the nucleotide sequence set forth in SEQ ID NO:83), SEQ ID NO:86 (which can be encoded by the nucleotide sequence set forth in SEQ ID NO:85), SEQ ID NO:88 (which can be encoded by the nucleotide sequence set forth in SEQ ID NO:87), SEQ ID NO:90 (which can be encoded by the nucleotide sequence set forth in SEQ ID NO:89), or SEQ ID NO:92 (which can be encoded by the nucleotide sequence set forth in SEQ ID NO:91).
[0093] In some aspects, the KAH polypeptide comprises a polypeptide having an amino acid sequence set forth in SEQ ID NO:94 (which can be encoded by the nucleotide sequence set forth in SEQ ID NO:93), SEQ ID NO:97 (which can be encoded by the nucleotide sequence set forth in SEQ ID NO:95 or SEQ ID NO:96), SEQ ID NO:100 (which can be encoded by the nucleotide sequence set forth in SEQ ID NO:98 or SEQ ID NO:99), SEQ ID NO:101, SEQ ID
NO:102, SEQ ID NO:103, SEQ ID NO:104, SEQ ID NO:106 (which can be encoded by the nucleotide sequence set forth in SEQ ID NO:105), SEQ ID NO:108 (which can be encoded by the nucleotide sequence set forth in SEQ ID NO:107), SEQ ID NO:110 (which can be encoded by the nucleotide sequence set forth in SEQ ID NO:109), SEQ ID NO:112 (which can be encoded by the nucleotide sequence set forth in SEQ ID NO:111), or SEQ ID
NO:114 (which can be encoded by the nucleotide sequence set forth in SEQ ID NO:113).
[0094] In some embodiments, a recombinant host comprises a nucleic acid encoding a UGT85C2 polypeptide (SEQ ID NO:7), a nucleic acid encoding a UGT76G1 polypeptide (SEQ
ID NO:9), a nucleic acid encoding a UGT74G1 polypeptide (SEQ ID NO:4), a nucleic acid encoding a UGT91D2 polypeptide, and/or a nucleic acid encoding a EUGT11 polypeptide (SEQ
ID NO:16). In some aspects, the UGT91D2 polypeptide can be a UGT91D2e polypeptide (SEQ
ID NO:11) or a UGT91D2e-b polypeptide (SEQ ID NO:13). In some aspects, the polypeptide can be encoded by the nucleotide sequence set forth in SEQ ID NO:5 or SEQ ID
NO:6, the UGT76G1 polypeptide can be encoded by the nucleotide sequence set forth in SEQ
ID NO:8, the UGT74G1 polypeptide can be encoded by the nucleotide sequence set forth in SEQ ID NO:3, the UGT91D2e polypeptide can be encoded by the nucleotide sequence set forth in SEQ ID NO:10, the UGT91D2e-b polypeptide can be encoded by the nucleotide sequence set forth in SEQ ID NO:12, and the EUGT11 polypeptide can be encoded by the nucleotide sequence set forth in SEQ ID NO:14 or SEQ ID NO:15. The skilled worker will appreciate that expression of these genes may be necessary to produce a particular steviol glycoside but that one or more of these genes can be endogenous to the host provided that at least one (and in some embodiments, all) of these genes is a recombinant gene introduced into the recombinant host. In a particular embodiment, a steviol-producing recombinant microorganism comprises exogenous nucleic acids encoding UGT85C2, UGT76G1, or UGT91D2 polypeptides.
[0095] In another particular embodiment, a steviol-producing recombinant microorganism comprises exogenous nucleic acids encoding UGT85C2, UGT76G1, UGT74G1, and polypeptides. In yet another particular embodiment, a steviol-producing recombinant microorganism comprises exogenous nucleic acids encoding UGT85C2, UGT76G1, UGT74G1, and EUGT11 polypeptides. In yet another particular embodiment, a steviol-producing recombinant microorganism comprises exogenous nucleic acids encoding UGT85C2, UGT76G1, UGT74G1, UGT91D2 (including inter alia UGT91D2e, UGT91D2m, UGT91D2e-b, and functional homologs thereof), and EUGT11 polypeptides. In yet another particular embodiment, a steviol-producing recombinant microorganism comprises exogenous nucleic acids encoding UGT85C2, UGT76G1, UGT74G1, UGT91D2, and/or EUGT11 polypeptides.
In yet another particular embodiment, a steviol-producing recombinant microorganism comprises exogenous nucleic acids encoding UGT85C2, UCT76G1, UGT74G1, UGT91D2, and/or EUGT11 polypeptides.
[0096] In some embodiments, a recombinant host comprises: (a) a gene encoding a polypeptide capable of beta 1,2 glucosylation of the 02 of the 19-0 glucose of a steviol glycoside; (b) a gene encoding a polypeptide capable of beta 1,2 glucosylation of the 02' of the 13-0-glucose of a steviol glycoside; (c) a gene encoding a polypeptide capable of beta 1,3 glucosylation of the 03' of the 19-0-glucose of a steviol glycoside; (d) a gene encoding a polypeptide capable of beta 1,3 glucosylation of the 03' of the 13-0-glucose of a steviol glycoside; (e) a gene encoding a polypeptide capable of beta 1,6 glucosylation of the 06' of the 13-0-glucose of a steviol glycoside; (f) a gene encoding a polypeptide capable of beta 1,6 glucosylation of the 06' of the 1,3-glucose of a 13-0 diglucoside moiety of a steviol glycoside;
(g) a gene encoding a polypeptide capable of glucosylation of the 13-0H of steviol or a steviol glycoside; (h) a gene encoding a polypeptide capable of glucosylation of the 0-19 carboxyl of steviol or a steviol glycoside; (i) a gene encoding a polypeptide capable of beta 1,2 rhamnosylation of the 02' of the 13-0-glucose of a steviol glycoside; (j) a gene encoding a polypeptide capable of beta 1,2 xylosylation of the 02' of the 13-0-glucose of a steviol glycoside; (o) a gene encoding a polypeptide capable of beta 1,2 GIcNAc transfer to the 02' of the 19-0 glucose of a steviol glycoside; (k) a gene encoding a polypeptide capable of beta 1,3 GIcNAc transfer to the 02' of the 19-0 glucose of a steviol glycoside; (I) a gene encoding a polypeptide capable of beta 1,3 GIcNAc transfer to the 02' of the 13-0-glucose of a steviol glycoside; (m) a gene encoding a polypeptide capable of GIcNAc transfer to the 0-19 carboxyl of steviol or a steviol glycoside; (n) a gene encoding a polypeptide capable of glucosylation of the 0-19 carboxyl of kaurenoic acid or kaurenol; (o) a gene encoding a polypeptide capable of beta 1,2 glucosylation of the 02 of the 19-0 glucose of a kaurenoic acid glycoside or kaurenol glycoside; (p) a gene encoding a polypeptide capable of a beta 1,2 glucosylation of a beta 1,2 diglucoside of kaurenoic acid; (q) a gene encoding a polypeptide capable of beta 1,2 GIcNAc transfer of a beta 1,2 diglucoside of kaurenoic acid; (r) a gene encoding a polypeptide capable of beta 1,3 glucosylation of the 03' of the 19-0-glucose of a kaurenoic acid glycoside or kaurenol glycoside; and/or (s) a gene encoding a polypeptide capable of beta 1,6 glucosylation of the 06' of the 1,3-glucose of a 19-0 diglucoside moiety of a steviol glycoside.
[0097] In some aspects, EUGT11 (SEQ ID NO:14/SEQ ID NO:15, SEQ ID NO:16), UGT91D2e (SEQ ID NO:10, SEQ ID NO:11), UGT91D2e-b (SEQ ID NO:12, SEQ ID
NO:13), a variant thereof, or a chimeric protein thereof catalyzes beta 1,2 glucosylation of the 02' of the 19-0 glucose of a steviol glycoside. Exemplary UGT91D2e variant sequences are set forth in SEQ ID NOs:1, 2, 118-121, 123, and 191-214. In some aspects, UGT91D2e (SEQ ID
NO:10, SEQ ID NO:11), UGT91D2e-b (SEQ ID NO:12, SEQ ID NO:13), a variant thereof, or a chimeric protein thereof catalyzes beta 1,2 glucosylation of the 02' of the 13-0-glucose of a steviol glycoside. Exemplary UGT91D2e variant sequences are set forth in SEQ ID NOs:1, 2, 118-121, 123, and 191-214. Exemplary UGT91D2e-EUGT11 chimeric protein sequences are set forth in SEQ ID NO:17 and SEQ ID NO:18. In some aspects, UGT76G1 (SEQ ID NO:8, SEQ ID
NO:9), a variant thereof, or a chimeric protein thereof catalyzes beta 1,3 glucosylation of the 03' of the 19-0-glucose of a steviol glycoside and/or beta 1,3 glucosylation of the 03' of the 13-0-glucose of a steviol glycoside. Exemplary UGT76G1 variant sequences are set forth in SEQ ID
NOs:181-190 and 217-220. In some aspects, UGT8502 (SEQ ID NO:5/SEQ ID NO:6, SEQ ID
NO:7), a variant thereof, or a chimeric protein thereof catalyzes glucosylation of the 13-0H of steviol or a steviol glycoside. Exemplary UGT8502 variant sequences are set forth in SEQ ID
NOs:127 and 147-180. In some aspects, UGT74G1 (SEQ ID NO:3, SEQ ID NO:4), a variant thereof, or a chimeric protein thereof catalyzes glucosylation of the 0-19 carboxyl of steviol or a steviol glycoside. In some aspects, EUGT11 (SEQ ID NO:14/SEQ ID NO:15, SEQ ID
NO:16), UGT91D2e (SEQ ID NO:10, SEQ ID NO:11), UGT74G1 (SEQ ID NO:3, SEQ ID NO:4), and/or UGT76G1 (SEQ ID NO:8, SEQ ID NO:9 can accept uridine diphosphate N-acetylglucosamine (UDP-Glc-NAc) as a substrate. In some aspects, UGT74G1 glycosylates ent-kaurenol and ent-kaurenoic acid; UGT76G1 and UGT91D2e subsequently add additional glucose or GIcNAc moieties by either a 1,3- or 1,2-linkage to form tri-glycosylated compounds.
See Figures 3, 7 and 8.
[0098] In some embodiments, steviol glycosides and/or steviol glycoside precursors are produced through contact of a steviol glycoside precursor with one or more enzymes involved in the steviol glycoside pathway in vitro. For example, contacting steviol with a UGT polypeptide can result in production of a steviol glycoside in vitro. In some embodiments, a steviol glycoside precursor is produced through contact of an upstream steviol glycoside precursor with one or more enzymes involved in the steviol glycoside pathway in vitro. For example, contacting ent-kaurenoic acid with a KAH enzyme can result in production of steviol in vitro.
[0099] In some embodiments, a steviol glycoside or steviol glycoside precursor is produced by whole cell bioconversion. For whole cell bioconversion to occur, a host cell expressing one or more enzymes involved in the steviol glycoside pathway takes up and modifies a steviol glycoside precursor in the cell; following modification in vivo, a steviol glycoside remains in the cell and/or is excreted into the culture medium. For example, a host cell expressing a gene encoding a UGT polypeptide can take up steviol and glycosylate steviol in the cell; following glycosylation in vivo, a steviol glycoside can be excreted into the culture medium. In some embodiments, the cell is permeabilized to take up a substrate to be modified or to excrete a modified product.
[00100] In some embodiments, steviol, one or more steviol glycoside precursors, and/or one or more steviol glycosides are produced by co-culturing of two or more hosts.
In some embodiments, one or more hosts, each expressing one or more enzymes involved in the steviol glycoside pathway, produce steviol, one or more steviol glycoside precursors, and/or one or more steviol glycosides. For example, a host comprising a GGPPS, a CDPS, a KO, a KS, a KAH, and/or a CPR and a host comprising one or more UGTs produce one or more steviol glycosides.
[00101] In some embodiments, polypeptides suitable for producing steviol glycosides, such as 1,2-stevioside and RebD, in vitro, in a recombinant host, or by whole cell bioconversion include functional homologs of UGT91D2e (SEQ ID NO:10, SEQ ID NO:11), including UGT91D2e-b (SEQ ID NO:12, SEQ ID NO:13); UGT91D2e V286C (SEQ ID NO:1);
UGT91D2e G384W (SEQ ID NO:2); UGT91D2e L211M (SEQ ID NO:118); UGT91D2e L195G (SEQ ID
NO:119); UGT91D2e V196P (SEQ ID NO:120); UGT91D2e L211H (SEQ ID NO:121);
UGT91D2e L213E (SEQ ID NO:191); UGT91D2e 5221Y (SEQ ID NO:192); UGT91D2e E438H

(SEQ ID NO:193); UGT91D2e M152T (SEQ ID NO:194); UGT91D2e L211C (SEQ ID
NO:195);
UGT91D2e L1955 (SEQ ID NO:196); UGT91D2e L195V (SEQ ID NO:197); UGT91D2e V286S

(SEQ ID NO:198); UGT91D2e S221S (SEQ ID NO:199); UGT91D2e P93V M152G (SEQ ID

NO:200); UGT91D2e S991 (SEQ ID NO:201); UGT91D2e T144K P201P (SEQ ID NO:202);
UGT91D2e T144L (SEQ ID NO:203); UGT91D2e T144M (SEQ ID NO:204); UGT91D2e A148K

L211I (SEQ ID NO:205); UGT91D2e L195N (SEQ ID NO:206); UGT91D2e K199C (SEQ ID
NO:207); UGT91D2e L211M E426G A466V (SEQ ID NO:208); UGT91D2e L211T 13031 (SEQ

ID NO:209); UGT91D2e V286N (SEQ ID NO:210); UGT91D2e 51 14F V2865 (SEQ ID
NO:211);
UGT91D2e G384K (SEQ ID NO:212); UGT91D2e G384Y (SEQ ID NO:213); UGT91D2e E438M (SEQ ID NO:214); and UGT91D2e L195C (SEQ ID NO:123). See Example 3.
[00102] In some embodiments, a useful UGT91D2 homolog can have one or more amino acid substitutions at residues 195, 196, 211, 286, and 384. See Table 2. Non-limiting examples of useful UGT91D2e homologs include polypeptides having substitutions (with respect to SEQ
ID NO:11) at residue 93 (e.g., a valine at residue 93); 99 (e.g., an isoleucine at residue 99), 114 (e.g., a phenylalanine at residue 114); 144 (e.g., a lysine, leucine, or methionine at residue 144);
148 (e.g., a lysine at residue 148); 152 (e.g., a threonine at residue 152);
195 (e.g., a glycine, cysteine, serine, arginine, or valine at residue 195); 196 (e.g., a proline at residue 196); 199 (e.g., a cysteine at residue 199); 211 (e.g., a methionine, histidine, threonine, cysteine, or isoleucine at residue 211); 213 (e.g., a glutamic acid at 213); 221 (e.g., an isoleucine at residue 221); 286 (e.g., an alanine, cysteine, asparagine, or serine at residue 286); 384 (e.g., a tryptophan, lysine, or tyrosine at residue 384); 426 (e.g., a glycine at residue 426); 438 (e.g., a histidine or methionine at residue 438); or 466 (e.g., a valine at residue 466). See Example 3.
[00103] In some embodiments, UGT91D2e variants comprise silent mutations.
For example, in some embodiments, UGT91D2e variants comprise silent mutations at residues not limited to residue 130, residue 201, or residue 221. See Example 3.
[00104] In some embodiments, UGT91D2e variants not limited to UGT91D2e V286C
(SEQ
ID NO:1), UGT91D2e G384W (SEQ ID NO:2), UGT91D2e L195V (SEQ ID NO:197), UGT91D2e V286S (SEQ ID NO:198), UGT91D2e T144K P201P (SEQ ID NO:202), UGT91D2e L211T 11301 (SEQ ID NO:184), UGT91D2e S11F V2865 (SEQ ID NO:211), and UGT91D2e E438M (SEQ ID NO:214) are selective towards rubusoside, with preferential accumulation of 1,2-stevioside. In some embodiments, UGT91D2e variants not limited to UGTD1D2e M152G (SEQ ID NO:200), UGT91D2e S991 (SEQ ID NO:201), UGT91D2e T144L (SEQ ID
NO:203), UGT91D2e A148K L221I (SEQ ID NO:205), and UGT91D2e G384K (SEQ ID
NO:212) are selective towards RebA, with preferential accumulation of RebD. In some embodiments, UGT91D2e variants not limited to a UGT91D2e variant with a mutation at residue 211 (e.g., UGT91D2e L211M of SEQ ID NO:118) catalyze conversion of rubusoside to 1,2-stevioside and conversion of RebA to RebD, with preferential accumulation of 1,2-stevioside.
See Example 3 and Tables 2 and 3.
[00105] In some embodiments, polypeptides suitable for producing steviol glycosides, such as RebA, RebD, rubusoside, and/or 1,2-stevioside in a recombinant host include UGT91D2e-b-EUGT11 chimeric enzymes, such as Chim_3 (SEQ ID NO:17) or Chim_7 (SEQ ID
NO:18). See Example 4 and Table 5.
[00106] In some embodiments, Chim_7 (SEQ ID NO:18) more efficiently converts rubusoside to 1,2-stevioside, compared to EUGT11 and UGT91D2e. In some embodiments, Chim_7 (SEQ
ID NO:18) fully consumes a supplied amount of rubusoside. In some embodiments, Chim_7 (SEQ ID NO:18) demonstrates 1.75-fold higher activity towards RebA than UGT91D2e-b (SEQ
ID NO:12, SEQ ID NO:13). In some embodiments, Chim_3 (SEQ ID NO:17) selectively converts rubusoside to 1,2-stevioside. See Example 4 and Table 5.
[00107] In some embodiments, UGT91D2e-b-EUGT11 chimeric enzymes such as Chim_2 (SEQ ID NO:122); Chim_4 (SEQ ID NO:124); Chim_5 (SEQ ID NO:125); Chim_6 (SEQ
ID
NO:126); Chim_7 (SEQ ID NO:18); Chim_8 (SEQ ID NO:128); Chim_9 (SEQ ID
NO:129);
Chim_10 (SEQ ID NO:130); Chim_11 (SEQ ID NO:131); Chim_12 (SEQ ID NO:132);
Chim_13 (SEQ ID NO:133); Chim_14 (SEQ ID NO:134) are used to produce steviol glycosides and/or steviol glycoside precursors.
[00108] In some embodiments, a useful UGT85C2 homolog can have one or more amino acid substitutions at residues 21, 48, 49, 84, 86, 87, 91, 92, 95, 122, 304, and 334. See Table 7.
Non-limiting examples of useful UGT85C2 homologs include polypeptides having substitutions (with respect to SEQ ID NO:7) at residue 21 (e.g., a lysine, threonine, or valine at residue 21), 48 (e.g., a serine, histidine, tyrosine, arginine, glutamine, or tryptophan at residue 48), 49 (e.g., a valine at residue 49), 84 (e.g., a glycine, alanine, threonine, cysteine, proline, valine, or asparagine at residue 84), 86 (e.g., an arginine or glycine at residue 86); 87 (e.g., an histidine, proline, methionine or tyrosine at residue 87); 91 (e.g., an lysine, arginine, or threonine at residue 91); 92 (e.g., an phenylalanine, isoleucine, methionine, or lysine at residue 92); 122 (e.g., an serine at residue 122); 304 (e.g., a serine at residue 304); and 334 (e.g., an serine or methionine at residue 334). See SEQ ID NOs:127 and 147-180, Table 7A for UGT85C2 variants analyzed that preferentially catalyze conversion of 19-SMG over conversion of steviol, Table 7B
for UGT85C2 variants that preferentially catalyze conversion of steviol over conversion of 19-SMG, and Table 70 for additional UGT85C2 variants that catalyze conversion of 19-SMG and steviol. Also see Example 5.
[00109] In some embodiments, a steviol glycoside-producing S. cerevisiae strain comprising a recombinant gene encoding a Synechococcus sp. GGPPS polypeptide (SEQ ID
NO:19, SEQ
ID NO:20), a recombinant gene encoding a truncated Z. mays CDPS polypeptide (SEQ ID
NO:39, SEQ ID NO:40), a recombinant gene encoding an A. thaliana KS
polypeptide (SEQ ID
NO:51, SEQ ID NO:52), a recombinant gene encoding a recombinant S. rebaudiana KO
polypeptide (SEQ ID NO:59, SEQ ID NO:60), a recombinant gene encoding an A.
thaliana ATR2 polypeptide (SEQ ID NO:91, SEQ ID NO:92), a recombinant gene encoding an 0. sativa EUGT11 polypeptide (SEQ ID NO:14/SEQ ID NO:15, SEQ ID NO:16), a recombinant gene encoding an SrKAHe1 polypeptide (SEQ ID NO:93, SEQ ID NO:94), a recombinant gene encoding an S. rebaudiana CPR8 polypeptide (SEQ ID NO:85, SEQ ID NO:86), a recombinant gene encoding an S. rebaudiana UGT74G1 polypeptide (SEQ ID NO:3, SEQ ID NO:4), a recombinant gene encoding an S. rebaudiana UGT76G1 polypeptide (SEQ ID NO:8, SEQ ID
NO:9), a recombinant gene encoding an S. rebaudiana UGT91D2e polypeptide (SEQ
ID NO:10, SEQ ID NO:11), a recombinant KO gene encoded by the nucleotide sequence set forth in SEQ
ID NO:67 (corresponding to the amino acid sequence set forth in SEQ ID
NO:117), and a recombinant CPR1 gene encoding (SEQ ID NO:77, SEQ ID NO:78) accumulates ent-kaurenoic acid+2GIc (#7), ent-kaurenoic acid+3GIc (isomer 1), ent-kaurenoic acid+3GIc (isomer 2), 19-SMG, steviol, stevio1+2GIc (#23), and stevio1+3GIc (#34) but does not accumulate ent-kaurenol glycosides. See Example 6 and Figures 4A-4C.
[00110] In some embodiments, the 584V F485, F48H, F48Y, F48R, F48Q, F48T, F485, I49V, P86R, P86G, and F1225 variants of UGT85C2 are selective towards 19-SMG, compared to steviol (Table 7A). In some embodiments, the 584T, I87M I87P, I87Y, L91K, L91R, L91T, L92M, and I95K variants of UGT85C2 are selective towards steviol, compared to (Table 7B). In some embodiments, expression of UGT85C2 T3045 (SEQ ID NO:127) in a steviol glycoside-producing host increases accumulation of steviol glycosides, compared to a steviol glycoside-producing host not expressing UGT85C2 T3045 (SEQ ID NO:127).
See Example 5.
[00111] In some embodiments, cell lysates comprising UGT85C2 or a UGT85C2 variant show a preference for either steviol or 19-SMG for a substrate. In some aspects, using steviol as a substrate, the F48H, F48Y, F48T, I49V, 584A, and L92F UGT85C2 variants exhibit high activity during incubation periods of under 40 min, and the F48H, F48Y, F48T, and I49V
UGT85C2 variants exhibit high activity during incubation periods of over 40 min (Table 8A).
Using 19-SMG as a substrate, the F48H, F48Y, F48T, I49V, and 584A UGT85C2 variants exhibit high activity during incubation periods of under 40 min, and the F48H, I49V, S84A, S84V, L91K, and L92F UGT85C2 variants, as well as the wild-type UGT85C2, exhibit high activity during incubation periods of over 40 min (Table 8B). In some aspects, the L91K, L91R, and L92F UGT85C2 variants exhibit a high 13-SMG/rubusoside ratio, whereas the F48Y, F48T, P86G UGT85C2 variants exhibit a low 13-SMG/rubusoside ratio. See Example 7.
[00112] In some embodiments, a useful UGT76G1 homolog can have one or more amino acid substitutions at residues 23, 26, 55, 146, 257, 283, and 337. See Example 4. Non-limiting examples of useful UGT76G1 homologs include polypeptides having substitutions (with respect to SEQ ID NO:9) at residue 21 (e.g., a lysine, threonine or valine at residue 21), residue 23 (e.g., a histidine at residue 23); residue 26 (e.g., a tryptophan at residue 26); residue 55 (e.g., a lysine at residue 55); residue 146 (e.g., a glycine at residue 146); residue 257 (e.g., a glycine at residue 257); residue 283 (e.g., a asparagine at residue 283); and residue 337 (e.g., a proline at residue 337). See SEQ ID NOs: 181-190. See Table 9 and Examples 8 and 9.
[00113] In some embodiments, expression of UGT76G1 variants that increase accumulation of RebD or RebM in steviol glycoside-producing S. cerevisiae strains (see WO
2014/122227, which has been incorporated by reference in its entirety) alter accumulation of 13-SMG, 1,2-bioside, rubusoside, RebA, RebB, RebD, RebE, RebM, RebG (1,3-stevioside), stevio1+3GIc (#1), stevio1+4GIc (#26), stevio1+5GIc (#22), stevio1+5GIc (#24), stevio1+5GIc (#25), stevio1+6GIc (isomer 1), and stevio1+6GIc (#23), compared to expression of wild-type UGT76G1 (SEQ ID
NO:9) in steviol glycoside-producing S. cerevisiae strains. See Figures 6, 10, 11D, and 11E and Examples 8 and 9.
[00114] In some embodiments, expression of UGT variants that increase RebD
levels in S.
cerevisiae also results in increased accumulation of stevio1+5GIc (#22), 1,2-stevioside, stevio1+6GIc (isomer 1), and stevio1+3GIc (#1) but decreased accumulation of stevio1+4GIc (#26), stevio1+5GIc (#24), and RebG (1,3-stevioside). In some embodiments, expression of UGT76G1 H155L (SEQ ID NO:184) results in increased accumulation of stevio1+5GIc (#25) but decreased accumulation of 1,2-stevioside, stevio1+3GIc (#1), stevio1+4GIc (#26), stevio1+5GIc (#22), stevio1+6GIc (isomer 1), and stevio1+6GIc (#23). In some embodiments, expression of UGT76G1 S253W (SEQ ID NO:186) results in decreased accumulation of 1,2-stevioside and stevio1+6GIc (isomer 1). In some embodiments, expression of UGT76G1 284G
results in increased accumulation of 1,2-stevioside and stevio1+6GIc (isomer 1) but decreased accumulation of RebG, stevio1+4GIc (#26), stevio1+5GIc (#25), and stevio1+6GIc (#23). See Figure 10 and Example 8.
[00115] In some embodiments, expression of UGT76G1 Q23H (SEQ ID NO:181), I26W (SEQ ID NO:182), UGT76G1 T146G (SEQ ID NO:183), UGT76G1 H155L (SEQ ID
NO:184), UGT76G1 L257G (SEQ ID NO:185), and UGT76G1 5283N (SEQ ID NO:188) decrease accumulation of stevio1+4GIc (#26). In some embodiments, expression of UGT76G1 Q23H (SEQ ID NO:181), UGT76G1 I26W (SEQ ID NO:182), UGT76G1 T146G (SEQ ID
NO:183), UGT76G1 L257G (SEQ ID NO:185), or UGT76G1 5283N (SEQ ID NO:188), all of which increase production of RebD, decrease accumulation of stevio1+5GIc (#25), compared to a control strain expressing wild-type UGT76G1. In some embodiments, expression of UGT76G1 H155L (SEQ ID NO:184), which increases RebM production, increases accumulation of stevio1+5GIc (#25). See Figure 11D and Example 8.
[00116] In some embodiments, expression of UGT76G1 Q23H (SEQ ID NO:181), I26W (SEQ ID NO:182), UGT76G1 T146G (SEQ ID NO:183), UGT76G1 L257G (SEQ ID
NO:185), or UGT76G1 5283N (SEQ ID NO:188) increases accumulation of stevio1+6GIc (#23), compared to a control strain expressing wild-type UGT76G1. In some embodiments, expression of UGT76G1 H155L (SEQ ID NO:184) decreases accumulation of stevio1+6GIc (#23). In some embodiments, expression of UGT76G1 Q23H (SEQ ID NO:181), I26W (SEQ ID NO:182), UGT76G1 T146G (SEQ ID NO:183), UGT76G1 L257G (SEQ ID
NO:185), or UGT76G1 5283N (SEQ ID NO:188) increases accumulation of stevio1+7GIc (isomer 2), compared to a control strain expressing wild-type UGT76G1. In some embodiments, expression of UGT76G1 H155L (SEQ ID NO:184) decreases accumulation of stevio1+7GIc (isomer 2). In some embodiments, expression of UGT76G1 Q23H (SEQ ID NO:181), UGT76G1 I26W (SEQ ID NO:182), UGT76G1 T146G (SEQ ID NO:183), UGT76G1 L257G
(SEQ ID NO:185), or UGT76G1 5283N (SEQ ID NO:188) increases accumulation of stevio1+7GIc (isomer 5). See Figure 11D and Example 8.
[00117] In some embodiments, a host expressing a gene encoding a UGT variant or UGT
chimeric polypeptide produces an increased level of glycosylated ent-kaurenoic acid and/or ent-kaurenol relative to a host not expressing a gene encoding a UGT variant or UGT chimeric polypeptide. In some embodiments, the UGT variant or UGT chimeric polypeptide comprises a UGT91D2e variant, a gene encoding a UGT91D2e-b-EUGT11 chimeric polypeptide, a gene encoding a UGT85C2 variant, and/or a gene encoding a UGT76G1 variant.
[00118] In some embodiments, a host expressing a gene encoding a UGT variant or UGT
chimeric polypeptide produces a decreased level of glycosylated ent-kaurenoic acid and/or ent-kaurenol relative to a host not expressing a gene encoding a UGT variant or UGT chimeric polypeptide. In some embodiments, the UGT variant or UGT chimeric polypeptide comprises a UGT91D2e variant, a gene encoding a UGT91D2e-b-EUGT11 chimeric polypeptide, a gene encoding a UGT85C2 variant, and/or a gene encoding a UGT76G1 variant.
[00119] In some embodiments, levels of ent-kaurenoic acid+2GIc (#7), ent-kaurenoic acid+3GIc (isomer 1), ent-kaurenoic acid+3GIc (isomer 2), ent-kaureno1+2GIc (#8), and ent-kaureno1+3G1c (isomer 1) co-eluted with ent-kaureno1+3GIc (#6) are altered in steviol glycoside-producing S. cerevisiae strains expressing wild-type UGT76G1 (SEQ ID NO:9), compared to S.
cerevisiae strains expressing UGT76G1 Q23H (SEQ ID NO:181), UGT76G1 I26W (SEQ
ID
NO:182), UGT76G1 T146G (SEQ ID NO:183), UGT76G1 H155L (SEQ ID NO:184), UGT76G1 L257G (SEQ ID NO:185), UGT76G1 S253W (SEQ ID NO:186), UGT76G1 T284G (SEQ ID
NO:187), UGT76G1 5283N (SEQ ID NO:188), UGT76G1 K337P (SEQ ID NO:189), or UGT76G1 T55K (SEQ ID NO:190). See Figure 9, Figures 11A-11C, and Example 8.
[00120] In some embodiments, S. cerevisiae strains expressing UGT76G1 variants that increase RebD levels also increase accumulation of ent-kaurenoic acid+2GIc (#7) and ent-kaurenoic acid+2GIc (isomer 1) but decrease accumulation of ent-kaurenoic acid+3GIc (isomer 2), compared to an S. cerevisiae strain expressing wild-type UGT76G1. In some embodiments, UGT76G1 variants that increase RebD levels also increase accumulation of ent-kaureno1+2GIc (#8) but decrease accumulation of ent-kaureno1+3GIc (isomer 1) co-eluted with ent-kaureno1+3G1c (#6). In some embodiments, expression of UGT76G1 H155L (SEQ ID
NO:184), a variant that increases levels of RebM, decreases accumulation of ent-kaurenoic acid+2GIc (#7) and ent-kaurenoic acid+3GIc (isomer 1). See Figure 9 and Example 8.
[00121] In some embodiments, total levels of glycosylated ent-kaurenoic acid (ent-kaurenoic acid+2GIc (#7) + ent-kaurenoic acid+3GIc (isomer 1) + ent-kaurenoic acid+3GIc (isomer 2)) are increased in steviol glycoside-producing S. cerevisiae strains expressing UGT76G1 Q23H (SEQ
ID NO:181), UGT76G1 I26W (SEQ ID NO:182), and UGT L257G (SEQ ID NO:185). In some embodiments, total levels of glycosylated ent-kaurenol (ent-kaureno1+3GIc (isomer 1) co-eluted with ent-kaureno1+3GIc (#6) and ent-kaureno1+2GIc (#8) are altered for in steviol glycoside-producing S. cerevisiae strains expressing UGT76G1 Q23H (SEQ ID NO:181), (SEQ ID NO:182), and UGT76G1 T146G (SEQ ID NO:183). See Figures 11B and 110 and Example 8.
[00122] In some embodiments, UGT variants not limited to variants of UGT76G1, UGT85C2, and/or UGT91D2e alter ratios of steviol glycosides produced to GIcNAc compounds and isomers thereof produced in vitro, in vivo in a host, and/or by whole cell bioconversion.

Exemplary GIcNAc structures include ent-kaurenoic acid+2GIc+1 GIcNAc and stevio1+4G1c+1GIcNAc (#11). See, e.g., Figures 7A, 7D, 8G-8I, and 8AC-8AF and Examples 6, 8, and 9.
[00123] In some embodiments, a steviol glycoside or steviol glycoside precursor composition produced in vivo, in vitro, or by whole cell bioconversion comprises fewer contaminants or less of any particular contaminant than a stevia extract from, inter alia, a stevia plant. Contaminants can include plant-derived compounds that contribute to off-flavors. Potential contaminants include pigments, lipids, proteins, phenolics, saccharides, spathulenol and other sesquiterpenes, labdane diterpenes, monoterpenes, decanoic acid, 8,11,14-eicosatrienoic acid, 2-methyloctadecane, pentacosane, octacosane, tetracosane, octadecanol, stigmasterol, 8-sitosterol, a-amyrin, 8-amyrin, lupeol, 8-amryin acetate, pentacyclic triterpenes, centauredin, quercitin, epi-alpha-cadinol, carophyllenes and derivatives, beta-pinene, beta-sitosterol, and gibberellins.
[00124] As used herein, the terms "detectable amount," "detectable concentration,"
"measurable amount," and "measurable concentration" refer to a level of steviol glycosides measured in area-under-curve (AUC), pM/0D600, mg/L, pM, or mM.
Steviol glycoside production (i.e., total, supernatant, and/or intracellular steviol glycoside levels) can be detected and/or analyzed by techniques generally available to one skilled in the art, for example, but not limited to, liquid chromatography-mass spectrometry (LC-MS), thin layer chromatography (TLC), high-performance liquid chromatography (HPLC), ultraviolet-visible spectroscopy/
spectrophotometry (UV-Vis), mass spectrometry (MS), and nuclear magnetic resonance spectroscopy (NMR).
[00125] As used herein, the term "undetectable concentration" refers to a level of a compound that is too low to be measured and/or analyzed by techniques such as TLC, HPLC, UV-Vis, MS, or NMR. In some embodiments, a compound of an "undetectable concentration" is not present in a steviol glycoside or steviol glycoside precursor composition.
[00126] As used herein, the terms "or" and "and/or" is utilized to describe multiple components in combination or exclusive of one another. For example, "x, y, and/or z" can refer to "x" alone, "y" alone, "z" alone, "x, y, and z," "(x and y) or z," "x or (y and z)," or "x or y or z." In some embodiments, "and/or" is used to refer to the exogenous nucleic acids that a recombinant cell comprises, wherein a recombinant cell comprises one or more exogenous nucleic acids selected from a group. In some embodiments, "and/or" is used to refer to production of steviol glycosides and/or steviol glycoside precursors. In some embodiments, "and/or"
is used to refer to production of steviol glycosides, wherein one or more steviol glycosides are produced. In some embodiments, "and/or" is used to refer to production of steviol glycosides, wherein one or more steviol glycosides are produced through one or more of the following steps: culturing a recombinant microorganism, synthesizing one or more steviol glycosides in a recombinant microorganism, and/or isolating one or more steviol glycosides.
Functional Homologs
[00127] Functional homologs of the polypeptides described above are also suitable for use in producing steviol glycosides in a recombinant host. A functional homolog is a polypeptide that has sequence similarity to a reference polypeptide, and that carries out one or more of the biochemical or physiological function(s) of the reference polypeptide. A
functional homolog and the reference polypeptide can be a natural occurring polypeptide, and the sequence similarity can be due to convergent or divergent evolutionary events. As such, functional homologs are sometimes designated in the literature as homologs, or orthologs, or paralogs.
Variants of a naturally occurring functional homolog, such as polypeptides encoded by mutants of a wild type coding sequence, can themselves be functional homologs. Functional homologs can also be created via site-directed mutagenesis of the coding sequence for a polypeptide, or by combining domains from the coding sequences for different naturally-occurring polypeptides ("domain swapping"). Techniques for modifying genes encoding functional polypeptides described herein are known and include, inter alia, directed evolution techniques, site-directed mutagenesis techniques and random mutagenesis techniques, and can be useful to increase specific activity of a polypeptide, alter substrate specificity, alter expression levels, alter subcellular location, or modify polypeptide-polypeptide interactions in a desired manner. Such modified polypeptides are considered functional homologs. The term "functional homolog" is sometimes applied to the nucleic acid that encodes a functionally homologous polypeptide.
[00128] Functional homologs can be identified by analysis of nucleotide and polypeptide sequence alignments. For example, performing a query on a database of nucleotide or polypeptide sequences can identify homologs of steviol glycoside biosynthesis polypeptides.
Sequence analysis can involve BLAST, Reciprocal BLAST, or PSI-BLAST analysis of non-redundant databases using a UGT amino acid sequence as the reference sequence.
Amino acid sequence is, in some instances, deduced from the nucleotide sequence.
Those polypeptides in the database that have greater than 40% sequence identity are candidates for further evaluation for suitability as a steviol glycoside biosynthesis polypeptide. Amino acid sequence similarity allows for conservative amino acid substitutions, such as substitution of one hydrophobic residue for another or substitution of one polar residue for another. If desired, manual inspection of such candidates can be carried out in order to narrow the number of candidates to be further evaluated. Manual inspection can be performed by selecting those candidates that appear to have domains present in steviol glycoside biosynthesis polypeptides, e.g., conserved functional domains. In some embodiments, nucleic acids and polypeptides are identified from transcriptome data based on expression levels rather than by using BLAST
analysis.
[00129] Conserved regions can be identified by locating a region within the primary amino acid sequence of a steviol glycoside biosynthesis polypeptide that is a repeated sequence, forms some secondary structure (e.g., helices and beta sheets), establishes positively or negatively charged domains, or represents a protein motif or domain. See, e.g., the Pfam web site describing consensus sequences for a variety of protein motifs and domains on the World Wide Web at sanger.ac.uk/Software/Pfam/ and pfam.janelia.org/. The information included at the Pfam database is described in Sonnhammer et al., Nucl. Acids Res., 26:320-322 (1998);
Sonnhammer etal., Proteins, 28:405-420 (1997); and Bateman etal., Nucl. Acids Res., 27:260-262 (1999). Conserved regions also can be determined by aligning sequences of the same or related polypeptides from closely related species. Closely related species preferably are from the same family. In some embodiments, alignment of sequences from two different species is adequate to identify such homologs.
[00130] Typically, polypeptides that exhibit at least about 40% amino acid sequence identity are useful to identify conserved regions. Conserved regions of related polypeptides exhibit at least 45% amino acid sequence identity (e.g., at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% amino acid sequence identity). In some embodiments, a conserved region exhibits at least 92%, 94%, 96%, 98%, or 99% amino acid sequence identity.
[00131] For example, polypeptides suitable for producing steviol in a recombinant host include functional homologs of UGTs.
[00132] Methods to modify the substrate specificity of, for example, a UGT, are known to those skilled in the art, and include without limitation site-directed/rational mutagenesis approaches, random directed evolution approaches and combinations in which random mutagenesis/saturation techniques are performed near the active site of the enzyme. For example see Osmani et al., 2009, Phytochemistry 70: 325-347.
[00133] A candidate sequence typically has a length that is from 80% to 200%
of the length of the reference sequence, e.g., 82, 85, 87, 89, 90, 93, 95, 97, 99, 100, 105, 110, 115, 120, 130, 140, 150, 160, 170, 180, 190, or 200% of the length of the reference sequence.
A functional homolog polypeptide typically has a length that is from 95% to 105% of the length of the reference sequence, e.g., 90, 93, 95, 97, 99, 100, 105, 110, 115, or 120% of the length of the reference sequence, or any range between. A `)/0 identity for any candidate nucleic acid or polypeptide relative to a reference nucleic acid or polypeptide can be determined as follows. A
reference sequence (e.g., a nucleic acid sequence or an amino acid sequence described herein) is aligned to one or more candidate sequences using the computer program Clustal Omega (version 1.2.1, default parameters), which allows alignments of nucleic acid or polypeptide sequences to be carried out across their entire length (global alignment). Chenna etal., 2003, Nucleic Acids Res. 31(13):3497-500.
[00134] Clustal Omega calculates the best match between a reference and one or more candidate sequences, and aligns them so that identities, similarities and differences can be determined. Gaps of one or more residues can be inserted into a reference sequence, a candidate sequence, or both, to maximize sequence alignments. For fast pairwise alignment of nucleic acid sequences, the following default parameters are used: word size:
2; window size: 4;
scoring method: %age; number of top diagonals: 4; and gap penalty: 5. For multiple alignment of nucleic acid sequences, the following parameters are used: gap opening penalty: 10.0; gap extension penalty: 5.0; and weight transitions: yes. For fast pairwise alignment of protein sequences, the following parameters are used: word size: 1; window size: 5;
scoring method: /0age; number of top diagonals: 5; gap penalty: 3. For multiple alignment of protein sequences, the following parameters are used: weight matrix: blosum; gap opening penalty:
10.0; gap extension penalty: 0.05; hydrophilic gaps: on; hydrophilic residues:
Gly, Pro, Ser, Asn, Asp, Gln, Glu, Arg, and Lys; residue-specific gap penalties: on. The Clustal Omega output is a sequence alignment that reflects the relationship between sequences. Clustal Omega can be run, for example, at the Baylor College of Medicine Search Launcher site on the World Wide Web (searchlauncher.bcm.tmc.edu/multi-align/multi-align.html) and at the European Bioinformatics Institute site at http://www.ebi.ac.uk/Tools/msa/clustalo/.
[00135] To determine a `)/0 identity of a candidate nucleic acid or amino acid sequence to a reference sequence, the sequences are aligned using Clustal Omega, the number of identical matches in the alignment is divided by the length of the reference sequence, and the result is multiplied by 100. It is noted that the% identity value can be rounded to the nearest tenth. For example, 78.11, 78.12, 78.13, and 78.14 are rounded down to 78.1, while 78.15, 78.16, 78.17, 78.18, and 78.19 are rounded up to 78.2.
[00136] It will be appreciated that functional UGT proteins can include additional amino acids that are not involved in the enzymatic activities carried out by the enzymes.
In some embodiments, UGT proteins are fusion proteins. The terms "chimera," "fusion polypeptide,"
"fusion protein," "fusion enzyme," "fusion construct," "chimeric protein,"
"chimeric polypeptide,"
"chimeric construct," and "chimeric enzyme" can be used interchangeably herein to refer to proteins engineered through the joining of two or more genes that code for different proteins. In some embodiments, a nucleic acid sequence encoding a UGT polypeptide can include a tag sequence that encodes a "tag" designed to facilitate subsequent manipulation (e.g., to facilitate purification or detection), secretion, or localization of the encoded polypeptide. Tag sequences can be inserted in the nucleic acid sequence encoding the polypeptide such that the encoded tag is located at either the carboxyl or amino terminus of the polypeptide.
Non-limiting examples of encoded tags include green fluorescent protein (GFP), human influenza hemagglutinin (HA), glutathione S transferase (GST), polyhistidine-tag (HIS tag), and Flag TM tag (Kodak, New Haven, CT). Other examples of tags include a chloroplast transit peptide, a mitochondrial transit peptide, an amyloplast peptide, signal peptide, or a secretion tag.
[00137] In some embodiments, a fusion protein is a protein altered by domain swapping. As used herein, the term "domain swapping" is used to describe the process of replacing a domain of a first protein with a domain of a second protein. In some embodiments, the domain of the first protein and the domain of the second protein are functionally identical or functionally similar. In some embodiments, the structure and/or sequence of the domain of the second protein differs from the structure and/or sequence of the domain of the first protein. In some embodiments, a UGT polypeptide is altered by domain swapping.
Steviol and Steviol Glycoside Biosynthesis Nucleic Acids
[00138] A recombinant gene encoding a polypeptide described herein comprises the coding sequence for that polypeptide, operably linked in sense orientation to one or more regulatory regions suitable for expressing the polypeptide. Because many microorganisms are capable of expressing multiple gene products from a polycistronic mRNA, multiple polypeptides can be expressed under the control of a single regulatory region for those microorganisms, if desired.
A coding sequence and a regulatory region are considered to be operably linked when the regulatory region and coding sequence are positioned so that the regulatory region is effective for regulating transcription or translation of the sequence. Typically, the translation initiation site of the translational reading frame of the coding sequence is positioned between one and about fifty nucleotides downstream of the regulatory region for a monocistronic gene.
[00139] In many cases, the coding sequence for a polypeptide described herein is identified in a species other than the recombinant host, i.e., is a heterologous nucleic acid. Thus, if the recombinant host is a microorganism, the coding sequence can be from other prokaryotic or eukaryotic microorganisms, from plants or from animals. In some case, however, the coding sequence is a sequence that is native to the host and is being reintroduced into that organism.
A native sequence can often be distinguished from the naturally occurring sequence by the presence of non-natural sequences linked to the exogenous nucleic acid, e.g., non-native regulatory sequences flanking a native sequence in a recombinant nucleic acid construct. In addition, stably transformed exogenous nucleic acids typically are integrated at positions other than the position where the native sequence is found. "Regulatory region"
refers to a nucleic acid having nucleotide sequences that influence transcription or translation initiation and rate, and stability and/or mobility of a transcription or translation product.
Regulatory regions include, without limitation, promoter sequences, enhancer sequences, response elements, protein recognition sites, inducible elements, protein binding sequences, 5' and 3' untranslated regions (UTRs), transcriptional start sites, termination sequences, polyadenylation sequences, introns, and combinations thereof. A regulatory region typically comprises at least a core (basal) promoter. A regulatory region also may include at least one control element, such as an enhancer sequence, an upstream element or an upstream activation region (UAR).
A
regulatory region is operably linked to a coding sequence by positioning the regulatory region and the coding sequence so that the regulatory region is effective for regulating transcription or translation of the sequence. For example, to operably link a coding sequence and a promoter sequence, the translation initiation site of the translational reading frame of the coding sequence is typically positioned between one and about fifty nucleotides downstream of the promoter. A
regulatory region can, however, be positioned as much as about 5,000 nucleotides upstream of the translation initiation site, or about 2,000 nucleotides upstream of the transcription start site.
[00140] The choice of regulatory regions to be included depends upon several factors, including, but not limited to, efficiency, selectability, inducibility, desired expression level, and preferential expression during certain culture stages. It is a routine matter for one of skill in the art to modulate the expression of a coding sequence by appropriately selecting and positioning regulatory regions relative to the coding sequence. It will be understood that more than one regulatory region may be present, e.g., introns, enhancers, upstream activation regions, transcription terminators, and inducible elements.
[00141] One or more genes can be combined in a recombinant nucleic acid construct in "modules" useful for a discrete aspect of steviol and/or steviol glycoside production. Combining a plurality of genes in a module, particularly a polycistronic module, facilitates the use of the module in a variety of species. For example, a steviol biosynthesis gene cluster, or a UGT gene cluster, can be combined in a polycistronic module such that, after insertion of a suitable regulatory region, the module can be introduced into a wide variety of species. As another example, a UGT gene cluster can be combined such that each UGT coding sequence is operably linked to a separate regulatory region, to form a UGT module. Such a module can be used in those species for which monocistronic expression is necessary or desirable. In addition to genes useful for steviol or steviol glycoside production, a recombinant construct typically also contains an origin of replication, and one or more selectable markers for maintenance of the construct in appropriate species.
[00142] It will be appreciated that because of the degeneracy of the genetic code, a number of nucleic acids can encode a particular polypeptide; i.e., for many amino acids, there is more than one nucleotide triplet that serves as the codon for the amino acid. Thus, codons in the coding sequence for a given polypeptide can be modified such that optimal expression in a particular host is obtained, using appropriate codon bias tables for that host (e.g., microorganism). As isolated nucleic acids, these modified sequences can exist as purified molecules and can be incorporated into a vector or a virus for use in constructing modules for recombinant nucleic acid constructs.
[00143] In some cases, it is desirable to inhibit one or more functions of an endogenous polypeptide in order to divert metabolic intermediates towards steviol or steviol glycoside biosynthesis. For example, it may be desirable to downregulate synthesis of sterols in a yeast strain in order to further increase steviol or steviol glycoside production, e.g., by downregulating squalene epoxidase. As another example, it may be desirable to inhibit degradative functions of certain endogenous gene products, e.g., glycohydrolases that remove glucose moieties from secondary metabolites or phosphatases as discussed herein. In such cases, a nucleic acid that overexpresses the polypeptide or gene product may be included in a recombinant construct that is transformed into the strain. Alternatively, mutagenesis can be used to generate mutants in genes for which it is desired to increase or enhance function.

Host Microorganisms
[00144] Recombinant hosts can be used to express polypeptides for the producing steviol glycosides. A number of prokaryotes and eukaryotes are suitable for use in constructing the recombinant microorganisms described herein, e.g., gram-negative bacteria, fungi (i.e., yeast), mammalian, insect, plant, and algae cells. A species and strain selected for use as a steviol glycoside production strain is first analyzed to determine which production genes are endogenous to the strain and which genes are not present. Genes for which an endogenous counterpart is not present in the strain are advantageously assembled in one or more recombinant constructs, which are then transformed into the strain in order to supply the missing function(s).
[00145] Typically, the recombinant microorganism is grown in a fermenter at a temperature(s) for a period of time, wherein the temperature and period of time facilitate the production of a steviol glycoside. The constructed and genetically engineered microorganisms provided by the invention can be cultivated using conventional fermentation processes, including, inter alia, chemostat, batch, fed-batch cultivations, semi-continuous fermentations such as draw and fill, continuous perfusion fermentation, and continuous perfusion cell culture.
Depending on the particular microorganism used in the method, other recombinant genes such as isopentenyl biosynthesis genes and terpene synthase and cyclase genes may also be present and expressed. Levels of substrates and intermediates, e.g., isopentenyl diphosphate, dimethylallyl diphosphate, GGPP, ent-kaurene and ent-kaurenoic acid, can be determined by extracting samples from culture media for analysis according to published methods.
[00146] Carbon sources of use in the instant method include any molecule that can be metabolized by the recombinant host cell to facilitate growth and/or production of the steviol glycosides. Examples of suitable carbon sources include, but are not limited to, sucrose (e.g., as found in molasses), fructose, xylose, ethanol, glycerol, glucose, cellulose, starch, cellobiose or other glucose-comprising polymer. In embodiments employing yeast as a host, for example, carbons sources such as sucrose, fructose, xylose, ethanol, glycerol, and glucose are suitable.
The carbon source can be provided to the host organism throughout the cultivation period or alternatively, the organism can be grown for a period of time in the presence of another energy source, e.g., protein, and then provided with a source of carbon only during the fed-batch phase.
[00147] After the recombinant microorganism has been grown in culture for the period of time, wherein the temperature and period of time facilitate the production of a steviol glycoside, steviol and/or one or more steviol glycosides can then be recovered from the culture using various techniques known in the art. In some embodiments, a permeabilizing agent can be added to aid the feedstock entering into the host and product getting out. For example, a crude lysate of the cultured microorganism can be centrifuged to obtain a supernatant. The resulting supernatant can then be applied to a chromatography column, e.g., a 0-18 column, and washed with water to remove hydrophilic compounds, followed by elution of the compound(s) of interest with a solvent such as methanol. The compound(s) can then be further purified by preparative HPLC. See also, WO 2009/140394.
[00148] It will be appreciated that the various genes and modules discussed herein can be present in two or more recombinant hosts rather than a single host. When a plurality of recombinant hosts is used, they can be grown in a mixed culture to accumulate steviol and/or steviol glycosides.
[00149] Alternatively, the two or more hosts each can be grown in a separate culture medium and the product of the first culture medium, e.g., steviol, can be introduced into second culture medium to be converted into a subsequent intermediate, or into an end product such as, for example, RebA. The product produced by the second, or final host is then recovered. It will also be appreciated that in some embodiments, a recombinant host is grown using nutrient sources other than a culture medium and utilizing a system other than a fermenter.
[00150] Exemplary prokaryotic and eukaryotic species are described in more detail below.
However, it will be appreciated that other species can be suitable. For example, suitable species can be in a genus such as Agaricus, Aspergifius, Bacillus, Candida, Corynebacterium, Eremothecium, Escherichia, Fusarium/Gibberella, Kluyveromyces, Laetiporus, Lentinus, Phaffia, Phanerochaete, Pichia, Physcomitrella, Rhodoturula, Saccharomyces, Schizosaccharomyces, Sphaceloma, Xanthophyllomyces or Yarrowia. Exemplary species from such genera include Lentinus tigrinus, Laetiporus sulphureus, Phanerochaete chrysosporium, Pichia pastoris, Cyberlindnera jadinfi, Physcomitrella patens, Rhodoturula glutinis, Rhodoturula mucilaginosa, Phaffia rhodozyma, Xanthophyllomyces dendrorhous, Fusarium fujikuroi/Gibberella fujikuroi, Candida utilis, Candida glabrata, Candida albicans, and Yarrowia lipolytica.
[00151] In some embodiments, a microorganism can be a prokaryote such as Escherichia bacteria cells, for example, Escherichia coli cells; Lactobacillus bacteria cells; Lactococcus bacteria cells; Comebacterium bacteria cells; Acetobacter bacteria cells;
Acinetobacter bacteria cells; or Pseudomonas bacterial cells.
[00152] In some embodiments, a microorganism can be an Ascomycete such as Gibberella fujikuroi, Kluyveromyces lactis, Schizosaccharomyces pombe, Aspergillus niger, Yarrowia lipolytica, Ashbya gossypfi, or S. cerevisiae.
[00153] In some embodiments, a microorganism can be an algal cell such as Blakeslea trispora, Dunaliefia sauna, Haematococcus pluvialis, Chloralla sp., Undaria pinnatifida, Sargassum, Laminaria japonica, Scenedesmus almeriensis species.
[00154] In some embodiments, a microorganism can be a cyanobacterial cell such as Blakeslea trispora, Dunaliella sauna, Haematococcus pluvialis, Chlorefia sp., Undaria pinnatifida, Sargassum, Laminaria japonica, Scenedesmus almeriensis.
Saccharomyces spp.
[00155] Saccharomyces is a widely used chassis organism in synthetic biology, and can be used as the recombinant microorganism platform. For example, there are libraries of mutants, plasmids, detailed computer models of metabolism and other information available for S.
cerevisiae, allowing for rational design of various modules to enhance product yield. Methods are known for making recombinant microorganisms.
Aspergifius spp.
[00156] Aspergifius species such as A. oryzae, A. niger and A. sojae are widely used microorganisms in food production and can also be used as the recombinant microorganism platform. Nucleotide sequences are available for genomes of A. nidulans, A.
fumigatus, A.
oryzae, A. clavatus, A. flavus, A. niger, and A. terreus, allowing rational design and modification of endogenous pathways to enhance flux and increase product yield. Metabolic models have been developed for Aspergifius, as well as transcriptomic studies and proteomics studies. A.
niger is cultured for the industrial production of a number of food ingredients such as citric acid and gluconic acid, and thus species such as A. niger are generally suitable for producing steviol glycosides.
E. coli
[00157] E. coli, another widely used platform organism in synthetic biology, can also be used as the recombinant microorganism platform. Similar to Saccharomyces, there are libraries of mutants, plasmids, detailed computer models of metabolism and other information available for E. coli, allowing for rational design of various modules to enhance product yield. Methods similar to those described above for Saccharomyces can be used to make recombinant E. coli microorganisms.
Agaricus, Gibberella, and Phanerochaete spp.
[00158] Agaricus, Gibberella, and Phanerochaete spp. can be useful because they are known to produce large amounts of isoprenoids in culture. Thus, the terpene precursors for producing large amounts of steviol glycosides are already produced by endogenous genes.
Thus, modules comprising recombinant genes for steviol glycoside biosynthesis polypeptides can be introduced into species from such genera without the necessity of introducing mevalonate or MEP pathway genes.
Arxula adeninivorans (Blastobotrys adeninivorans)
[00159] Arxula adeninivorans is dimorphic yeast (it grows as budding yeast like the baker's yeast up to a temperature of 42 C, above this threshold it grows in a filamentous form) with unusual biochemical characteristics. It can grow on a wide range of substrates and can assimilate nitrate. It has successfully been applied to the generation of strains that can produce natural plastics or the development of a biosensor for estrogens in environmental samples.
Yarrowia lipolytica
[00160] Yarrowia lipolytica is dimorphic yeast (see Arxula adeninivorans) and belongs to the family Hemiascomycetes. The entire genome of Yarrowia lipolytica is known.
Yarrowia species is aerobic and considered to be non-pathogenic. Yarrowia is efficient in using hydrophobic substrates (e.g. alkanes, fatty acids, oils) and can grow on sugars. It has a high potential for industrial applications and is an oleaginous microorgamism.
Yarrowia lipolyptica can accumulate lipid content to approximately 40% of its dry cell weight and is a model organism for lipid accumulation and remobilization. See e.g., Nicaud, 2012, Yeast 29(10):409-18; Beopoulos et al., 2009, Biochimie 91(6):692-6; Banker et al., 2009, App/ Microbiol Biotechnol. 84(5):847-65.
Rhodotorula sp.
[00161] Rhodotorula is unicellular, pigmented yeast. The oleaginous red yeast, Rhodotorula glutinis, has been shown to produce lipids and carotenoids from crude glycerol (Saenge et al., 2011, Process Biochemistry 46(1):210-8). Rhodotorula toruloides strains have been shown to be an efficient fed-batch fermentation system for improved biomass and lipid productivity (Li et al., 2007, Enzyme and Microbial Technology 41:312-7).
Rhodosporidium toruloides
[00162] Rhodosporidium toruloides is oleaginous yeast and useful for engineering lipid-production pathways (See e.g. Zhu et al., 2013, Nature Commun. 3:1112; Ageitos et al., 2011, Applied Microbiology and Biotechnology 90(4):1219-27).
Candida boidinii
[00163] Candida boidinii is methylotrophic yeast (it can grow on methanol).
Like other methylotrophic species such as Hansenula polymorpha and Pichia pastoris, it provides an excellent platform for producing heterologous proteins. Yields in a multigram range of a secreted foreign protein have been reported. A computational method, IPRO, recently predicted mutations that experimentally switched the cofactor specificity of Candida boidinii xylose reductase from NADPH to NADH. See, e.g., Mattanovich et al., 2012, Methods Mol Biol.
824:329-58; Khoury et al., 2009, Protein Sci. 18(10):2125-38.
Hansenula polymorpha (Pichia angusta)
[00164] Hansenula polymorpha is methylotrophic yeast (see Candida boidinii).
It can furthermore grow on a wide range of other substrates; it is thermo-tolerant and can assimilate nitrate (see also Kluyveromyces lactis). It has been applied to producing hepatitis B vaccines, insulin and interferon alpha-2a for the treatment of hepatitis C, furthermore to a range of technical enzymes. See, e.g., Xu et al., 2014, Virol Sin. 29(6):403-9.
Kluyveromyces lactis
[00165] Kluyveromyces lactis is yeast regularly applied to the production of kefir. It can grow on several sugars, most importantly on lactose which is present in milk and whey. It has successfully been applied among others for producing chymosin (an enzyme that is usually present in the stomach of calves) for producing cheese. Production takes place in fermenters on a 40,000 L scale. See, e.g., van Ooyen et al., 2006, FEMS Yeast Res. 6(3):381-92.
Pichia pastoris
[00166] Pichia pastoris is methylotrophic yeast (see Candida boidinii and Hansenula polymorpha). It provides an efficient platform for producing foreign proteins.
Platform elements are available as a kit and it is worldwide used in academia for producing proteins. Strains have been engineered that can produce complex human N-glycan (yeast glycans are similar but not identical to those found in humans). See, e.g., Piirainen etal., 2014, N
Biotechnol. 31(6):532-7.
Physcomitrella spp.
[00167] Physcomitrella mosses, when grown in suspension culture, have characteristics similar to yeast or other fungal cultures. This genera can be used for producing plant secondary metabolites, which can be difficult to produce in other types of cells.
Steviol Glycoside Compositions
[00168] Steviol glycosides do not necessarily have equivalent performance in different food systems. It is therefore desirable to have the ability to direct the synthesis to steviol glycoside compositions of choice. Recombinant hosts described herein can produce compositions that are selectively enriched for specific steviol glycosides (e.g., RebD or RebM) and have a consistent taste profile. As used herein, the term "enriched" is used to describe a steviol glycoside composition with an increased proportion of a particular steviol glycoside, compared to a steviol glycoside composition (extract) from a stevia plant. Thus, the recombinant hosts described herein can facilitate the production of compositions that are tailored to meet the sweetening profile desired for a given food product and that have a proportion of each steviol glycoside that is consistent from batch to batch. In some embodiments, hosts described herein do not produce or produce a reduced amount of undesired plant by-products found in Stevia extracts. Thus, steviol glycoside compositions produced by the recombinant hosts described herein are distinguishable from compositions derived from Stevie plants.
[00169] It will be appreciated that the amount of an individual steviol glycoside (e.g., RebA, RebB, RebD, or RebM) produced by the recombinant host cell disclosed herein can accumulate in the cell culture broth from about Ito about 7,000 mg/L, e.g., about Ito about 10 mg/L, about 3 to about 10 mg/L, about 5 to about 20 mg/L, about 10 to about 50 mg/L, about 10 to about 100 mg/L, about 25 to about 500 mg/L, about 100 to about 1,500 mg/L, or about 200 to about 1,000 mg/L, at least about 1,000 mg/L, at least about 1,200 mg/L, at least about at least 1,400 mg/L, at least about 1,600 mg/L, at least about 1,800 mg/L, at least about 2,800 mg/L, or at least about 7,000 mg/L. In some aspects, the amount of an individual steviol glycoside produced by the recombinant host cell disclosed herein can exceed 7,000 mg/L in the cell culture broth.
[00170] It will be appreciated that the amount of a combination of steviol glycosides (e.g., RebA, RebB, RebD, or RebM) produced by the recombinant host cell disclosed herein can accumulate in the cell culture broth from about 1 mg/L to about 7,000 mg/L, e.g., about 200 to about 1,500, at least about 2,000 mg/L, at least about 3,000 mg/L, at least about 4,000 mg/L, at least about 5,000 mg/L, at least about 6,000 mg/L, or at least about 7,000 mg/L. In some aspects, the amount of a combination of steviol glycosides produced by the recombinant host cell disclosed herein can exceed 7,000 mg/L. In general, longer culture times will lead to greater amounts of product. Thus, the recombinant microorganism can be cultured for from 1 day to 7 days, from 1 day to 5 days, from 3 days to 5 days, about 3 days, about 4 days, or about days.
[00171] It will be appreciated that the various genes and modules discussed herein can be present in two or more recombinant microorganisms rather than a single microorganism. When a plurality of recombinant microorganisms is used, they can be grown in a mixed culture to produce steviol and/or steviol glycosides. For example, a first microorganism can comprise one or more biosynthesis genes for producing a steviol glycoside precursor, while a second microorganism comprises steviol glycoside biosynthesis genes. The product produced by the second, or final microorganism is then recovered. It will also be appreciated that in some embodiments, a recombinant microorganism is grown using nutrient sources other than a culture medium and utilizing a system other than a fermenter.
[00172] Alternatively, the two or more microorganisms each can be grown in a separate culture medium and the product of the first culture medium, e.g., steviol, can be introduced into second culture medium to be converted into a subsequent intermediate, or into an end product such as RebA. The product produced by the second, or final microorganism is then recovered.
It will also be appreciated that in some embodiments, a recombinant microorganism is grown using nutrient sources other than a culture medium and utilizing a system other than a ferm enter.
[00173] Steviol glycosides and compositions obtained by the methods disclosed herein can be used to make food products, dietary supplements and sweetener compositions.
See, e.g., WO 2011/153378, WO 2013/022989, WO 2014/122227, and WO 2014/122328.
[00174] For example, substantially pure steviol or steviol glycoside such as RebM or RebD
can be included in food products such as ice cream, carbonated beverages, fruit juices, yogurts, baked goods, chewing gums, hard and soft candies, and sauces. Substantially pure steviol or steviol glycoside can also be included in non-food products such as pharmaceutical products, medicinal products, dietary supplements and nutritional supplements.
Substantially pure steviol or steviol glycosides may also be included in animal feed products for both the agriculture industry and the companion animal industry. Alternatively, a mixture of steviol and/or steviol glycosides can be made by culturing recombinant microorganisms separately, each producing a specific steviol or steviol glycoside, recovering the steviol or steviol glycoside in substantially pure form from each microorganism and then combining the compounds to obtain a mixture comprising each compound in the desired proportion. The recombinant microorganisms described herein permit more precise and consistent mixtures to be obtained compared to current Stevia products.
[00175] In another alternative, a substantially pure steviol or steviol glycoside can be incorporated into a food product along with other sweeteners, e.g. saccharin, dextrose, sucrose, fructose, erythritol, aspartame, sucralose, monatin, or acesulfame potassium.
The weight ratio of steviol or steviol glycoside relative to other sweeteners can be varied as desired to achieve a satisfactory taste in the final food product.
See, e.g., U.S. 2007/0128311. In some embodiments, the steviol or steviol glycoside may be provided with a flavor (e.g., citrus) as a flavor modulator.
[00176] Compositions produced by a recombinant microorganism described herein can be incorporated into food products. For example, a steviol glycoside composition produced by a recombinant microorganism can be incorporated into a food product in an amount ranging from about 20 mg steviol glycoside/kg food product to about 1800 mg steviol glycoside/kg food product on a dry weight basis, depending on the type of steviol glycoside and food product. For example, a steviol glycoside composition produced by a recombinant microorganism can be incorporated into a dessert, cold confectionary (e.g., ice cream), dairy product (e.g., yogurt), or beverage (e.g., a carbonated beverage) such that the food product has a maximum of 500 mg steviol glycoside/kg food on a dry weight basis. A steviol glycoside composition produced by a recombinant microorganism can be incorporated into a baked good (e.g., a biscuit) such that the food product has a maximum of 300 mg steviol glycoside/kg food on a dry weight basis. A
steviol glycoside composition produced by a recombinant microorganism can be incorporated into a sauce (e.g., chocolate syrup) or vegetable product (e.g., pickles) such that the food product has a maximum of 1000 mg steviol glycoside/kg food on a dry weight basis. A steviol glycoside composition produced by a recombinant microorganism can be incorporated into bread such that the food product has a maximum of 160 mg steviol glycoside/kg food on a dry weight basis. A steviol glycoside composition produced by a recombinant microorganism, plant, or plant cell can be incorporated into a hard or soft candy such that the food product has a maximum of 1600 mg steviol glycoside/kg food on a dry weight basis. A steviol glycoside composition produced by a recombinant microorganism, plant, or plant cell can be incorporated into a processed fruit product (e.g., fruit juices, fruit filling, jams, and jellies) such that the food product has a maximum of 1000 mg steviol glycoside/kg food on a dry weight basis. In some embodiments, a steviol glycoside composition produced herein is a component of a pharmaceutical composition.
See, e.g., Steviol Glycosides Chemical and Technical Assessment 69th JECFA, 2007, prepared by Harriet Wallin, Food Agric. Org.;
EFSA Panel on Food Additives and Nutrient Sources added to Food (ANS), "Scientific Opinion on the safety of steviol glycosides for the proposed uses as a food additive," 2010, EFSA
Journal 8(4):1537;
U.S. Food and Drug Administration GRAS Notice 323; U.S Food and Drug Administration GRAS Notice Notice 329; WO 2011/037959; WO 2010/146463; WO 2011/046423; and WO

2011/056834.
[00177] For example, such a steviol glycoside composition can have from 90-99 weight `)/0 RebA and an undetectable amount of stevia plant-derived contaminants, and be incorporated into a food product at from 25-1600 mg/kg, e.g., 100-500 mg/kg, 25-100 mg/kg, mg/kg, 50-500 mg/kg or 500-1000 mg/kg on a dry weight basis.
[00178] Such a steviol glycoside composition can be a RebB-enriched composition having greater than 3 weight % RebB and be incorporated into the food product such that the amount of RebB in the product is from 25-1600 mg/kg, e.g., 100-500 mg/kg, 25-100 mg/kg, 250-1000 mg/kg, 50-500 mg/kg or 500-1000 mg/kg on a dry weight basis. Typically, the RebB-enriched composition has an undetectable amount of stevia plant-derived contaminants.
[00179] Such a steviol glycoside composition can be a RebD-enriched composition having greater than 3 weight % RebD and be incorporated into the food product such that the amount of RebD in the product is from 25-1600 mg/kg, e.g., 100-500 mg/kg, 25-100 mg/kg, 250-1000 mg/kg, 50-500 mg/kg or 500-1000 mg/kg on a dry weight basis. Typically, the RebD-enriched composition has an undetectable amount of stevia plant-derived contaminants.
[00180] Such a steviol glycoside composition can be a RebE-enriched composition having greater than 3 weight % RebE and be incorporated into the food product such that the amount of RebE in the product is from 25-1600 mg/kg, e.g., 100-500 mg/kg, 25-100 mg/kg, 250-1000 mg/kg, 50-500 mg/kg or 500-1000 mg/kg on a dry weight basis. Typically, the RebE-enriched composition has an undetectable amount of stevia plant-derived contaminants.
[00181] Such a steviol glycoside composition can be a RebM-enriched composition having greater than 3 weight % RebM and be incorporated into the food product such that the amount of RebM in the product is from 25-1600 mg/kg, e.g., 100-500 mg/kg, 25-100 mg/kg, 250-1000 mg/kg, 50-500 mg/kg or 500-1000 mg/kg on a dry weight basis. Typically, the RebM-enriched composition has an undetectable amount of stevia plant-derived contaminants.
[00182] In some embodiments, a substantially pure steviol or steviol glycoside is incorporated into a tabletop sweetener or "cup-for-cup" product. Such products typically are diluted to the appropriate sweetness level with one or more bulking agents, e.g., maltodextrins, known to those skilled in the art. Steviol glycoside compositions enriched for RebA, RebB, RebD, RebE, or RebM, can be package in a sachet, for example, at from 10,000 to 30,000 mg steviol glycoside/kg product on a dry weight basis, for tabletop use. In some embodiments, a steviol glycoside produced in vitro, in vivo, or by whole cell bioconversion
[00183] The invention will be further described in the following examples, which do not limit the scope of the invention described in the claims.
EXAMPLES
[00184] The Examples that follow are illustrative of specific embodiments of the invention, and various uses thereof. They are set forth for explanatory purposes only, and are not to be taken as limiting the invention.
Example 1: LC-MS Analytical Procedures
[00185] LC-MS analyses for Examples 3 and 4 were performed using an Agilent 1200 Series HPLC system (Agilent Technologies) fitted with a Phenomenex Kinetex 018 column (150 x 2.1 mm, 2.6 pm particles, 100 A pore size) connected to a TSQ Quantum Access (ThermoFisher Scientific) triple quadropole mass spectrometer with a heated electrospray ion (HESI) source.
Elution was carried out using a mobile phase of eluent B (MeCN with 0.1%
Formic acid) and eluent A (water with 0.1% Formic acid) by increasing the gradient from 10-40%
B from min 0.0 to 1.0, increasing 40-50% B in min 1.0 to 6.5, and increasing 50-100% B from min 6.5 to 7Ø

The flow rate was 0.4 mL/min, and the column temperature was 30 C. 1,2-stevioside and RebD
were detected using SIM (Single Ion Monitoring) in positive mode.
[00186] LC-MS analyses for Examples 8 and 9 were performed on Waters ACQUITY
UPLCO
(Waters Corporation) with a Waters ACQUITY UPLCO BEH C18 column (2.1 x 50 mm, 1.7 pm particles, 130 A pore size) equipped with a pre-column (2.1 x 5 mm, 1.7 pm particles, 130 A
pore size) coupled to a Waters ACQUITY TQD triple quadropole mass spectrometer with electrospray ionization (ESI) operated in negative ionization mode. Compound separation was achieved using a gradient of the two mobile phases: A (water with 0.1% formic acid) and B
(MeCN with 0.1% formic acid) by increasing from 20% to 50 % B between 0.3 to 2.0 min, increasing to 100% B at 2.01 min, holding 100% B for 0.6 min, and re-equilibrating for 0.6 min.
The flow rate was 0.6 mL/min, and the column temperature was set at 55 C.
Steviol glycosides were monitored using SIM (Single Ion Monitoring) and quantified by comparing against authentic standards. See Table 1 for m/z trace and retention time values of steviol glycosides detected.
Table 1: LC-MS Analytical Data for Steviol and Steviol Glycosides Compound MS RT Fig ure(s) Table(s) Trace (min) stevio1+5GIc (#22) 1127.48 0.85 6D, 7E, 9C, 9F, 91 [also referred to as compound 5.22] 8AK-8AN, 10A, 10B, stevio1+6GIc (isomer 1) 1289.53 0.87 6D, 7B, 8M- 9C, 9F, [also referred to as compound 6.1] 8P, 10A, 10B, 11D
stevio1+7GIc (isomer 2) 1451.581 0.94 6D, 7B, 8Q- 9C, 9F, [also referred to as compound 7.2] 8T, 11D
stevio1+6GIc (#23) 1289.53 0.97 6D, 10A, 9F, 91 [also referred to as compound 6.23] 10B, 11D
RebE 965.42 1.06 6B, 6C, 10C, 9A, 9D, RebD 1127.48 1.08 6A, 6C, 10C, 2, 3, 5, 11E 9A, 9D, RebM 1289.53 1.15 6A, 6C, 10C, 9A, 9D, stevio1+7GIc (isomer 5) 1451.581 1.09 7C, 8Y-8AB, 9F, 91 [also referred to as compound 7.5] 11D
stevio1+7GIc (#13) 1451.581 0.94 6D

Compound MS RT
Figure(s) Table(s) Trace (min) [also referred to as compound 7.13]
stevio1+4G1c (#26) 965.42 1.21 6D, 7D, 90, 9F, [also referred to as compound 4.26] 8AG-8AJ, 9H
10A, 10B, stevio1+4G1c (#33) 965.42 1.49 90, 91 [also referred to as compound 4.33]
stevio1+5G1c (#24) 1127.48 1.18 6D, 10A, 9F, 91 [also referred to as compound 5.24] 10B, 11D
stevio1+4G1c (#25) 1127.48 1.40 6D, 10A, 5,90, 9F, [also referred to as compound 5.25] 10B, 11D 91 RebA 965.42 1.43 6A, 60, 100, 9A, 9D, Rebl 1127.48 1.4 9H
1,2-stevioside 803.37 1.43 10B, 11D 2, 3, 5, 9B, 9E, stevio1+3G1c (#1) 803.37 1.52 6D, 10A, 9B, 9E
[also referred to as compound 3.1] 10B, 11D
stevio1+2G1c (#23) 641.32 1.57 40 [also referred to as compound 2.23]
stevio1+3G1c (#34) 803.37 40 90, 9E
[also referred to as compound 3.34]
RebQ 965.42 1.59 1,3-stevioside (RebG) 803.37 1.60 6B-6D, 10B, 9D, 9G

rubusoside 641.32 1.67 5, 6B, 60, 5, 8B, 80, 10C, 11E 9D, 9G
RebB 803.37 1.76 6A, 60, 100, 9A, 9D, 1,2-bioside 641.32 1.80 6B-D, 100, 9A, 9D, 11D, 11E 9G
1,3-bioside 641.32 1.95 9E
13-SMG 479.26 2.04 4B, 6A, 60, 8A, 8B, 10C, 11E 80,9A, 9D, 9G
19-SMG 525.27 1.98 4B 7A, 7B, 70, 8B, 80, 9E, Compound MS RT
Figure(s) Table(s) Trace (min) ent-kaurenoic acid+3GIc (isomer 1) 787.37 2.16 4A, 7A, 8A- 9B, 9E, [also referred to as compound KA3.1] 80, 9A, 11A, 9H

ent-kaurenoic acid+3GIc (isomer 2) 787.37 2.28 4A, 7A, 8D- 9B, 9E, [also referred to as compound KA3.2] 8F, 9A, 11A, 9H

ent-kaureno1+3GIc (isomer 1) 773.4 2.36 4A, 7A, 8J-co-eluted with ent-kaureno1+3GIc (#6) 8L, 9B, 11A, [also referred to as compounds KL3.1 110 and KL3.6]
ent-kaurenoic acid+2GIc (#7) 625.32 2.35 4A, 9A, 11A, 9B, 9D, [also referred to as compound KA2.7] 11B 9H
ent-kaureno1+2GIc (#8) 611.34 2.38 9B, 7B, 11A, 9B, 9E
[also referred to as compound KL2.8] 110 Steviol 317.21 2.39 40 7A, 7B, 70, 8A, 8B, 80,
[00187]
Steviol glycosides, including GIcNAc-derivatives, glycosylated ent-kaurenol, and/or glycosylated ent-kaurenoic acid can be isolated using a method described herein. For example, following fermentation, a culture broth can be centrifuged for 30 min at 7000 rpm at 4 C to remove cells, or cells can be removed by filtration. The cell-free lysate can be obtained, for example, by mechanical disruption or enzymatic disruption of the host cells and additional centrifugation to remove cell debris. Mechanical disruption of the dried broth materials can also be performed, such as by sonication. The dissolved or suspended broth materials can be filtered using a micron or sub-micron prior to further purification, such as by preparative chromatography. The fermentation media or cell-free lysate can optionally be treated to remove low molecular weight compounds such as salt; and can optionally be dried prior to purification and re-dissolved in a mixture of water and solvent. The supernatant or cell-free lysate can be purified as follows: a column can be filled with, for example, HP20 Diaion0 resin (Supelco) or other suitable non-polar adsorbent or reverse phase chromatography resin, and an aliquot of supernatant or cell-free lysate can be loaded on to the column and washed with water to remove the hydrophilic components. The steviol glycoside product can be eluted by stepwise incremental increases in the solvent concentration in water or a gradient from, a g., 0% ¨>
100% methanol).
The levels of steviol glycosides, glycosylated ent-kaurenol, and/or glycosylated ent-kaurenoic acid in each fraction, including the flow-through, can then be analyzed by LC-MS. Fractions can then be combined and reduced in volume using a vacuum evaporator. Additional purification steps can be utilized, if desired, such as additional chromatography steps and crystallization.
Example 2: Strain Engineering and Fermentation
[00188] Steviol glycoside-producing S. cerevisiae strains were constructed as described in WO 2011/153378, WO 2013/022989, WO 2014/122227, and WO 2014/122328, each of which is incorporated by reference in their entirety. For example, a yeast strain comprising one or more copies of a recombinant gene encoding a Synechococcus sp. GGPPS
polypeptide (SEQ
ID NO:19, SEQ ID NO:20), a recombinant gene encoding a truncated Z. mays CDPS
polypeptide (SEQ ID NO:39, SEQ ID NO:40), a recombinant gene encoding an A.
thaliana KS
polypeptide (SEQ ID NO:51, SEQ ID NO:52), a recombinant gene encoding a recombinant S.
rebaudiana KO polypeptide (SEQ ID NO:59, SEQ ID NO:60), a recombinant gene encoding an A. thaliana ATR2 polypeptide (SEQ ID NO:91, SEQ ID NO:92), a recombinant gene encoding an 0. sativa EUGT11 polypeptide (SEQ ID NO:14/SEQ ID NO:15, SEQ ID NO:16), a recombinant gene encoding an SrKAHe1 polypeptide (SEQ ID NO:93, SEQ ID NO:94), a recombinant gene encoding an S. rebaudiana CPR8 polypeptide (SEQ ID NO:85, SEQ
ID
NO:86), a recombinant gene encoding an S. rebaudiana UGT85C2 polypeptide (SEQ
ID
NO:5/SEQ ID NO:6, SEQ ID NO:7) or a UGT85C2 variant (or functional homolog) of SEQ ID
NO:7, a recombinant gene encoding an S. rebaudiana UGT74G1 polypeptide (SEQ ID
NO:3, SEQ ID NO:4) or a UGT74G1 variant (or functional homolog) of SEQ ID NO:4, a recombinant gene encoding an S. rebaudiana UGT76G1 polypeptide (SEQ ID NO:8, SEQ ID NO:9) or a UGT76G1 variant (or functional homolog) of SEQ ID NO:9, and a recombinant gene encoding an S. rebaudiana UGT91D2e polypeptide (SEQ ID NO:10, SEQ ID NO:11) or a UGT91D2e variant (or functional homolog) of SEQ ID NO:11 such as a UGT91D2e-b (SEQ ID
NO:12, SEQ
ID NO:13) polypeptide produced steviol glycosides.
Example 3: Modulation of Substrate-Specificity of UGT91D2e
[00189] UGT91D1 (GenBank Accession No. AY345980) is highly expressed in the Stevia plant and thought to be a functional UGT. However, its substrate is not a steviol glycoside. This suggests that UGT91D1 has a different substrate than UGT91D2e, which may be defined by the 22 amino acids with which it differs from UGT91D2e. A UGT91D2e site saturation library (SSL) screen of the 22 amino acids differing from UGT91D1 was prepared using Geneart (Life Technologies) and degenerate NNK-primers.
[00190] UGT91D2 SSL clones were expressed in E. coli XJb (DE3) Autolysism"
cells (Zymo Research). Colonies were grown overnight in 96 deep-well plates at 37 C with 1 mL NZCYM
(pH 7.0) comprising 15 g Tryptone, 7.5 g NaCI, 7.5 g yeast extract, 1.5 g casamino acids, 3 g MgSO4 and fortified with 100 mg/L ampicillin and 33 mg/L chloramphenicol. 150 pL overnight cultures were transferred to 24 deep-well plates comprising 3 mL NZCYM with ampicillin, 0.1 mM isopropyl-3-D-1-thiogalactopyranoside (IPTG), 3 mM L-arabinose, and 2%
(v/v) ethanol and incubated 20 h at 20 C. Cells were pelleted and lysed in 100 pL lysis buffer (10 mM Tris-HCI pH
8.0, 5 mM MgC12, 1 mM CaCl2, 3 tablets/100 mL Complete mini protease inhibitor cocktail (Roche)) by a single freeze-thaw cycle and 50 pL DNase mix (1 pL 1.4 mg/mL
deoxyribonuclease (Calbiochem), 1.2 pL 500 mM MgC12, and 47.8 pL of 4x PBS
buffer). Plates were shaken at 500 rpm for 5 min at 25 C to allow degradation of genomic DNA.
Plates were then spun down at 4000 rpm for 30 min at 4 C. See WO 2013/022989, which is incorporated by reference in its entirety.
[00191] Activity of UGT91D2e variants was tested in vitro to assess the specificity of the UGT91D2e variants towards the substrates, rubusoside and RebA. 6 pL of the lysates were diluted with 24 pL of reaction mixture (final concentration: 100 mM Tris-HCI
(pH 8.0), 5 mM
MgC12, 1 mM KCI, 300 pM uridine diphosphate glucose (UDPG), and 100 pM
rubusoside or RebA). The reaction mixture was incubated at 30 C for 24 h, and 1,2-stevioside and RebD
production was measured by LC-MS. Results are shown in Table 2.
Table 2.
Activity of UGT91D2e-b and UGT91D2e variants on rubusoside and RebA, producing 1,2-stevioside and RebD, respectively.
1,2-stevioside (pM) RebD (pM) 1,2-stevioside/RebD
UGT91D2e-b 264.9 2.7 98.1 (SEQ ID NO:13) UGT91D2e V286C 59.3 0.0 N/A (No activity on (SEQ ID NO:1) RebA) UGT91D2e G384W 205.6 0.0 N/A (No activity on (SEQ ID NO:2) RebA) UGT91D2e L211M 129.7 3.7 35.1 (SEQ ID NO:118) UGT91D2e L195G 178.4 0.9 198.2 (SEQ ID NO:119) UGT91D2e V196P 162.1 2.4 67.5 (SEQ ID NO:120) UGT91D2e L211H 123.5 5.1 24.2 (SEQ ID NO:121)
[00192] As shown in Table 2, rubusoside and RebA were substrates of UGT91D2e-b (SEQ
ID NO:13), UGT91D2e L211M (SEQ ID NO:118), UGT91D2e L195G (SEQ ID NO:119), UGT91D2e V196P (SEQ ID NO:120), and UGT91D2e L211H (SEQ ID NO:121), as 1,2-stevioside and RebD were produced upon contact of the enzymes with either rubusoside or RebA. However, the ratio of 1,2-stevioside/RebD produced by UGT91D2e-b (SEQ ID
NO:13), UGT91D2e L211M (SEQ ID NO:118), UGT91D2e L195G (SEQ ID NO:119), UGT91D2e V196P

(SEQ ID NO:120), and UGT91D2e L211H (SEQ ID NO:121) fluctuated from 24.2 to 198.2, indicating that the enzymes were not equally selective towards either substrate. The UGT91D2e V286C and UGT91D2e G384W variants were selective towards rubusoside;
no RebD was produced upon contact of either variant with RebA.
[00193] Additional variants of UGT91D2e were found to demonstrate substrate specificity towards rubusoside or RebA using the above-described assay. See Table 3. The variants of SEQ ID NO:200 (P93V M152G), SEQ ID NO:201 (S991), SEQ ID NO:203 (T144L), SEQ
ID
NO:205 (A148K L2211), SEQ ID NO:212 (G384K) were selective towards RebA. The UGT91D2e variants of SEQ ID NO:197 (L195V), SEQ ID NO:198 (V2865), SEQ ID
NO:202 (T144K P201P (silent)), SEQ ID NO:209 (L211T 11301 (silent)), SEQ ID NO:211 (5114F V2865), SEQ ID NO:214 (E438M) were selective towards rubusoside.
Table 3. Activity of UGT91D2e variants on rubusoside and RebA, producing 1,2-stevioside and RebD, respectively.
Variant 1,2-stevioside (pM) RebD (pM) 1,2-stevioside/RebD
UGT91D2e L213E 13.6 1.1 12.4 (SEQ ID NO:191) UGT91D2e S221Y 13.1 27.1 0.5 (SEQ ID NO:192) UGT91D2e E438H 5.1 1.4 3.6 (SEQ ID NO:193) UGT91D2e M 152T 16.8 1.5 11.2 (SEQ ID NO:194) UGT91D2e L211C 7.3 1.6 15.8 (SEQ ID NO:195) UGT91D2e L195S 16.4 1.4 11.7 (SEQ ID NO:196) UGT91D2e L195V 35.9 0.0 N/A (No activity on (SEQ ID NO:197) RebA) UGT91D2e V286S 14.2 0.0 N/A (No activity on Variant 1,2-stevioside (pM) RebD (pM) 1,2-stevioside/RebD
(SEQ ID NO:198) RebA) UGT91D2e S221S 16.2 1.7 9.5 (silent) (SEQ ID NO:199) UGT91D2e P93V 0.2 2.5 0.1 (SEQ ID NO:200) UGT91D2e S99I 0.2 2.6 0.1 (SEQ ID NO:201) UGT91D2e T144K 1.6 0.0 N/A (No activity on P201P (silent) RebA) (SEQ ID NO:202) UGT91D2e T144L 0.0 2.6 0.0 (No activity on (SEQ ID NO:203) rubusoside) UGT91D2e T144M 1.3 1.6 0.8 (SEQ ID NO:204) UGT91D2e A148K 0.2 2.7 0.1 (SEQ ID NO:205) UGT91D2e L195N 5.1 1.0 5.1 (SEQ ID NO:206) UGT91D2e K199C 2.6 1.3 2.0 (SEQ ID NO:207) UGT91D2e L211M 79.1 1.1 71.9 (SEQ ID NO:208) UGT91D2e L21 IT 2.7 0.0 N/A (No activity on 13031 (silent) RebA) (SEQ ID NO:209) UGT91D2e V286N 3.0 0.0 N/A (No activity on (SEQ ID NO:210) RebA) UGT91D2e S1 14F 5.9 0.0 N/A (No activity on V2865 RebA) (SEQ ID NO:211) UGT91D2e G384K 0.0 2.2 0.0 (No activity on (SEQ ID NO:212) rubusoside) UGT91D2e G384Y 2.9 1.9 1.5 (SEQ ID NO:213) UGT91D2e E438M 4.7 0.0 N/A (No activity on (SEQ ID NO:214) RebA) UGT91D2e L195C 3.2 1.3 2.5 (SEQ ID NO:123) Example 4: Evaluation of UGT91D2e-b-EUGT11 Chimeric Enzymes
[00194] UGT91D2e-b-EUGT11 chimeric enzymes were tested in vitro to access activity on the substrates, rubusoside and RebA. UGT91D2e-b-EUGT11 chimeras were created by polymerase chain reaction (PCR)-amplification and overlap extension FOR using the primers in Table 4.
Table 4. Primers Used to Create UGT91D2e-b-EUGT11 Chimeric Enzymes.
Description Sequence SEQ ID
Vector (forward) GGCAAGCCACGTTTGGTG SEQ
ID NO:135 Vector (reverse) GGAGCTGCATGTGTCAGAGG SEQ
ID NO:136 EUGT11 fragment 1 /
CGATGTATTTCATCACTGGTTGCC SEQ ID NO:137 UGT91D2e-b fragment 2 ATCCATCGCGGCT
(forward) EUGT11 / UGT91D2e-b AGCCGCGATGGATGGCAACCAGT SEQ ID NO:138 fragment 2 (reverse) GAT GAAATACATCG
UGT91D2e-b fragment 1 /
TTATGATTATACTCACTACTGGGC SEQ ID NO:139 EUGT11 fragment 2 (forward) TGCTGCAGCCGCATTG
UGT91D2e-b fragment 1 /
AGCCGCGATGGATGGCAACCAGT SEQ ID NO:140 EUGT11 fragment 2 (reverse) GAT GAAATACATCG
EUGT11 fragment 2 /
CAAACCTATTACTTTCCTTGGTTT SEQ ID NO:141 UGT91D2e-b fragment 3 ACTGCCACCGGAAATAC
(forward) EUGT11 fragment 2/
GTATTTCCGGTGGCAGTAAACCA SEQ ID NO:142 UGT91D2e-b fragment 3 AGGAAAGTAATAGGTTTG
(reverse) UGT91D2e-b fragment 2 /
CCGGTGGTTCCGGTGGGACTAAT SEQ ID NO:143 EUGT11 fragment 3 (forward) GCCTCCATTACATGA
UGT91D2e-b fragment 2 /
TCATGTAATGGAGGCATTAGTCCC SEQ ID NO:144 EUGT11 fragment 3 (reverse) ACCGGAACCACCGG
EUGT11 fragment 3/
GAACGCAGGTCTGCAGGTTCCAA SEQ ID NO:145 UGT91D2e-b fragment 4 GAAATGAGGAAGATGG
(forward) EUGT11 fragment 3 /
CCATCTTCCTCATTTCTTGGAACC SEQ ID NO:146 UGT91D2e-b fragment 4 TGCAGACCTGCGTTC
(reverse)
[00195] UGT91D2e-b-EUGT11 chimeric enzymes were expressed in E. coli XJb(DE3) AutolysisTM cells (Zymo Research). Colonies were grown in 50 mL NZCYM (pH 7.0) with ampicillin and chloramphenicol and re-inoculated into 500 mL NZCYM with IPTG, L-arabinose, and ethanol. Cell lysate preparations were done in 15 mL lysis buffer followed by 150 pL
DNase and 200 pL 500 mM MgC12. GST-tag affinity purification of the chimeras was performed by adding 1/3 volume of 4x PBS buffer (560 mM NaCI, 10.8 mM KCI, 40 mM
Na2HPO4, 7.2 mM
KH2PO4 (PH 7.3)) to the lysate supernatant, followed by incubation (2 h, 4 C) with Glutathione Sepharose 4B (GE Healthcare) and loading onto Poly-Prep Chromatography Columns (Bio-Rad). The beads were washed twice with 1X PBS buffer and eluted with 50 mM
Tris-HCI (pH
8.0) and 10 mM reduced glutathione. Eluted protein was stabilized by addition of glycerol to a final concentration of 50%. SDS-PAGE was performed using NuPAGE 4-12 % Bis-Tris 1.0 mm precast gels (Invitrogen), NuPAGE MOPS (Invitrogen) running buffer and SimplyBlue SafeStain (Invitrogen). The amounts of chimeras produced were determined from the relative staining intensity of the gel images using ImageJ software.
[00196] Chimeras were screened by adding 20 pL purified UGT91D2e-b, EUGT11, or UGT91D2e-b-EUGT11 chimeric enzymes (0.02 mg/mL) to a total volume of 80 pL
reaction mixture comprising 100 mM Tris-HCI (pH 8.0), 5 mM MgC12, 1 mM KCI, 300 pM
uridine diphosphate glucose (UDPG), and 100 pM rubusoside or RebA. The reactions were incubated at 30 C for 24 h, and levels of RebA, RebD, rubusoside, and 1,2-stevioside were measured by LC-MS. Not all of the chimeras purified were active in the above described assay (see Table 5 for enzymes having activity on rubusoside and/or RebA).
Table 5. EUGT11, UGT91D2e-b, and EUGT11-UGT91D2e-b chimeric enzyme activity on RebA and rubusoside.
RebA (pM) RebD (pM) rubusoside 1,2-steviosi de (PM) (AUC) EUGT11 (SEQ 32.230 101.300 34.899 1188497 ID NO:16) UGT91D2e-b 97.314 6.580 41.157 2660570 (SEQ ID NO:13) Chim_3 (SEQ 109.764 NF 138.911 11435 ID NO:17) Chim_7 (SEQ 88.502 11.510 NF 3693895 ID NO:18) *NF=Not Found
[00197] As shown in Table 5, Chim_7 (SEQ ID NO:18) more efficiently converted rubusoside to 1,2-stevioside, compared to EUGT11 and UGT91D2e. Chim_7 (SEQ ID NO:18) fully consumed the supplied amount of rubusoside, unlike EUGT11 or UGT91D2e. When incubating EUGT11 with rubusoside, the 019-position of rubusoside was 1,2-glycosylated, and RebE and 1,2-stevioside were also produced (Table 5). Additionally, Chim_7 (SEQ ID
NO:18) demonstrated 1.75-fold higher activity towards RebA than UGT91D2e-b. Chim_3 (SEQ ID
NO:17) selectively converted rubusoside to 1,2-stevioside; no RebA was converted to RebD by Chim_3 (SEQ ID NO:17) (Table 5).
Example 5: Evaluation of UGT85C2 Variants
[00198] Three homology models of UGT85C2 were generated with the ORCHESTRA
module in Sybyl-X 2.0 (Certara) using a combination of the three PDB templates (Model 1:
2PQ6, 2VCE, 2C1X; Model 2: 2PQ6; Model 3: 2PQ6, 2C1X) and using standard settings and sequences for UGT85H2, UGT7261, and VvGT1 (see PDB2PQ6, PDB2VCE, and PCB2C1X).

Model geometry and quality were checked with the molprobity and ProQ
webservers (see Chen et al., Acta Crystallographica. Section D, Biological Crystallography 66(Pt 1):12-21 (2010), Davis et al., Nucleic Acids Research 35:W375-83 (2007), Wallner & Elofsson, Protein Science:
A Publication of the Protein Society 12(5):1073-86 (2003). The fluorinated UDPG sugar donor analog, UDP-2FGIc, from PDB:2VCE was imported into the UDPG binding site of prior to the acceptors steviol, 13-SMG, 19-SMG, or rubusoside. Steviol and steviol glycosides were prepared using the Sybyl-X small molecule builder and docked into the active site of the enzyme with the Surflex Dock suite using standard GeomX settings. The sites for the site saturation library (SSL) were determined by selecting all the residues within 3 A of the ligands in the docking analysis that were not 100% conserved in the PDB-templates. See Table 6.
Table 6. SSL residues for UGT85C2 Docking Analysis.
UGT85C2 Model UGT85C2 Model UGT85C2 Model Conserved #1 #2 #3 Phe18 Pro19 Ala20 GIn21 UGT85C2 Model UGT85C2 Model UGT85C2 Model Conserved #1 #2 #3 Ser22 x x x His23 x x x C
Lys25 x x Phe48 x x 11e49 x G1n52 x Glu82 x Ala83 x Ser84 x Pro86 x 11e87 x Arg88 x x Leu91 x x Leu92 x 11e95 x Phe122 x Thr143 x x Leu144 x x x Asp198 x Va1207 x Phe210 x Thr211 x Asn300 x Phe301 x C
G1y302 x x C
Ser303 x x Thr304 x x x Thr305 x x x Va1306 x Leu334 x Trp359 x C
G1n362 x C
His377 x x C
G1y379 x x C
Trp380 x x x C
Gly381 x x Ser382 x x x C
Tyr398 x x UGT85C2 Model UGT85C2 Model UGT85C2 Model Conserved #1 #2 #3 Trp400 Asp401 GIn402 x: Residue within 3 A of steviol, 19-SMG, and UDPG in the docking analysis C: Conserved residue
[00199] SSL clones were generated for the 34 non-conserved amino acids in Table 6 predicted to be within 3 A of the ligands residues. A modified version of the whole plasmid amplification method (Zheng etal. Nucleic Acids Research 32(14):e115 (2004)) was used with overlapping NNK-primers and Phusion polymerase. 10 pL PCR reaction was treated with 10 U
Dpnl (New England Biolabs) at 37 C for 1 h, heat inactivated at 65 C for 20 min, and transformed into E. colt DH5a cells. Colonies were selected on Luria Broth (LB) + kanamycin agar plates and grown in 4 mL LB fortified with kanamycin. Plasmids were purified using the GeneJETTIvl miniprep kit (Thermo Fisher Scientific) and sequenced.
[00200] The sequence-verified site saturation library (SSL) clones were transformed into E.
colt XJb(DE3) AutolysisTM cells (Zymo Research) and selected on LB + kanamycin agar plates.
Single colonies were inoculated into 1 mL NZCYM fortified with 30 mg/L
kanamycin and incubated overnight at 37 C and 200 rpm orbital shaking. 50 pL of the overnight culture were transferred into 1 mL of fresh NZCYM fortified with 30 mg/L kanamycin, 3 mM
arabinose, and 0.1 mM IPTG and incubated overnight at 20 C and 200 rpm orbital shaking. The cells were spun down at 3220 g /10 min at 4 C and resuspended in 50 pL GT-buffer (10 mM
Tris-HCI (pH
7.5), 5 mM MgC12, 1 mM CaCl2) comprising complete Mini EDTA free protease inhibitor cocktail (1 tablet / 25 mL GT-buffer; Roche Diagnostics). Pellets were resuspended by orbital shaking at 200 rpm / 5 min at 4 C. Cells were incubated at -80 C for minimum 15 min before initiation of lysing step.
[00201] The cells were lysed by heating the samples to 25 C and adding 25 pL
DNAse I mix comprising of 2.39 mL 4x His binding buffer (80 mM Tris-HCI (pH 7.5), 500 mM
NaCI, 10 mM
Imidazole) with 50 pL 1.4 mg/mL DNAse I bovine pancreas (Calbiochem) and 60 pL
MgC12 (500 mM). The lysates were filtered through a 1.2 pm 96-well filterplate (EMD
Millipore) and transferred to another 1.2 pm filterplate comprising 50 pL His-select beads (Sigma-Aldrich) prewashed twice with 1X binding buffer. The lysates and beads were then incubated for 2 h at 4 C with 500 rpm orbital shaking. The plates were spun down at 450 g / 2 min.
Total protein concentration in the flow-through was measured using the Bradford assay reagent (Sigma-Aldrich), the samples were washed twice by centrifuging the samples, removing supernatants and adding 50 pL 1X His binding buffer. Elution buffer (20 mM Tris-HCI (pH
7.5), 500 mM NaCI, 250 mM imidazole) was added to the beads and incubated for 5 min at 4 C at 500 rpm orbital shaking and the proteins eluted into a 96 well FOR plate (FrameStar 96, 4titude). The purifications were evaluated by running samples of the flow-through, washing steps and eluate on NuPAGEO SDS-PAGE gel system with 4-12% Bis-Tris precast gels (Invitrogen).
[00202] Activity of the purified UGT85C2 variants was measured. 2.0 pg/mL

variant was incubated for 20 min at 37 C with reaction buffer (100 mM Tris-HCI
(pH 8.0), 1 mM
KCI, Calf Intestinal Alkaline Phosphatase (New England Biolabs), 120 pM UDPG, and either 40 pM steviol or 40 pM 19-SMG). In this assay, the glucose on UDPG was transferred to steviol or 19-SMG; the products were UDP and either 13-SMG or rubusoside. The phosphates on UDP
were then released by a phosphatase, and the amount of phosphate released was measured at Abs600 using the Malachite green protocol (Baykov et al., Analytical Biochemistry 171(2):266-70). Values were normalized by total protein released measured by using Bradford reagent (Sigma-Aldrich).
[00203] Candidates were selected as having activity of one standard deviation or higher than wild-type activity or having less than 50% activity on one substrate while maintaining wild-type activity on the other (e.g., exhibiting substrate-specificity). The Abs600 ratios of a steviol sample to a 19-SMG sample for wild-type UGT85C2 (SEQ ID NO:7) averaged 0.94, indicating that the wild-type UGT85C2 catalyzes conversion of steviol and 19-SMG with little or no preference of substrate. Table 7A shows the UGT85C2 variants analyzed that preferentially catalyzed conversion of 19-SMG over conversion of steviol, Table 7B shows the UGT85C2 variants analyzed that preferentially catalyzed conversion of steviol over conversion of 19-SMG, and Table 70 shows the UGT85C2 variants analyzed that catalyzed conversion of 19-SMG and steviol with little preference for either substrate. Particular clones generated by the site saturation library (SSL) screen were selected more than once, corresponding to more than one entry in Tables 7A-C.
Table 7A. UGT85C2 SSL screen candidates that were selective towards 19-SMG
as a substrate.

Steviol 19-SMG Steviol/19- Sum Mutation UGT85C2 SMG Variant SEQ ID
(Abssoo) (Abssoo) Abssoo (Abssoo) Ratio 0.105 0.165 0.636 0.27 F48S SEQ ID NO:150 0.099 0.136 0.728 0.235 F48H SEQ ID NO:151 0.089 0.142 0.627 0.231 F48Y SEQ ID NO:152 0.080 0.117 0.684 0.197 F48R SEQ ID NO:153 0.068 0.126 0.540 0.194 F48Q SEQ ID NO:154 0.068 0.112 0.607 0.18 F48T SEQ ID NO:156 0.065 0.114 0.570 0.179 F485 SEQ ID NO:150 0.094 0.141 0.667 0.235 149V SEQ ID NO:157 0.078 0.111 0.703 0.189 149V SEQ ID NO:157 0.116 0.238 0.487 0.354 584V SEQ ID NO:164 -0.020 0.153 19-SMG 0.133 584V SEQ ID NO:164 0.096 0.230 0.417 0.326 P86R SEQ ID NO:165 0.083 0.196 0.423 0.279 P86R SEQ ID NO:165 0.065 0.17 0.382 0.235 P86R SEQ ID NO:165 0.042 0.18 0.233 0.222 P86G SEQ ID NO:166 -0.003 0.169 19-SMG 0.166 P86R SEQ ID NO:165 Table 7B. UGT85C2 SSL screen candidates that were selective towards steviol as a substrate.
Steviol 19-SMG Steviol/19- Sum Mutation UGT85C2 SMG Variant SEQ ID
(Abssoo) (Abssoo) Ratio (Abssoo) 0.382 -0.081 Steviol 0.301 584T SEQ ID NO:160 0.242 -0.083 Steviol 0.159 584T SEQ ID NO:160 0.521 -0.033 Steviol 0.488 I87M SEQ ID NO:169 0.261 0.190 1.374 0.451 187Y SEQ ID NO:170 0.372 0.159 2.340 0.531 L91K SEQ ID NO:171 0.369 0.134 2.754 0.503 L91K SEQ ID NO:171 0.228 0.104 2.192 0.332 L91R SEQ ID NO:172 0.202 0.079 2.557 0.281 L91R SEQ ID NO:172 0.147 0.041 3.585 0.188 L91T SEQ ID NO:173 0.606 0.266 2.278 0.872 195K SEQ ID NO:177 Table 7C. UGT85C2 SSL screen candidates that were not substrate selective towards steviol or 19-SMG.

Steviol 19-SMG Steviol/19- Sum Mutation UGT85C2 SMG Variant SEQ ID
(Abssoo) (Abssoo) Ratio (Abs600) 0.229 0.268 0.854 0.497 Q21L SEQ ID NO:147 0.231 0.261 0.885 0.492 Q21T SEQ ID NO:148 0.214 0.252 0.849 0.466 Q21V SEQ ID NO:149 0.083 0.098 0.847 0.181 F48W SEQ ID NO:155 0.359 0.332 1.081 0.691 584G SEQ ID NO:158 0.306 0.331 0.924 0.637 584A SEQ ID NO:159 0.296 0.292 1.014 0.588 S84C SEQ ID NO:161 0.250 0.299 0.836 0.549 584P SEQ ID NO:162 0.250 0.256 0.977 0.506 584A SEQ ID NO:159 0.219 0.262 0.836 0.481 584N SEQ ID NO:163 0.355 0.306 1.160 0.661 187H SEQ ID NO:167 0.326 0.274 1.190 0.600 187P SEQ ID NO:168 0.308 0.282 1.092 0.590 187M SEQ ID NO:169 0.279 0.216 1.292 0.495 187Y SEQ ID NO:170 0.474 0.426 1.113 0.900 L92F SEQ ID NO:174 0.387 0.331 1.169 0.718 L921 SEQ ID NO:175 0.342 0.260 1.315 0.602 L92M SEQ ID NO:176 0.39 0.598 0.652 0.988 F1225 SEQ ID NO:178 0.297 0.248 1.198 0.545 L3345 SEQ ID NO:179 0.27 0.233 1.159 0.503 L334M SEQ ID NO:180
[00204] The purified 584V and P86R variants of UGT85C2 were selective towards 19-SMG;
UGT85C2 584V and UGT85C2 P86R did not demonstrate activity on steviol (Table 7A). The purified F485, F48H, F48Y, F48R, F48Q, F48T, F485, I49V, P86R, P86G, and F1225 UGT85C2 variants also showed selectivity towards 19-SMG (Table 7A). However, the purified 584T and I87M variants of UGT85C2 were selective towards steviol; UGT85C2 584T
and UGT85C2 I87M did not demonstrate activity on 19-SMG (Table 7B). The purified I87P, I87Y, L91K, L91R, L91T, L92M, and I95K UGT85C2 variants also showed selectivity towards steviol (Table 7B).
Example 6: Characterization of Steviol Glycoside-Producing Yeast Strain Deleted of
[00205] A modified version of the steviol glycoside-producing S. cerevisiae strain described in Example 2, a recombinant KO gene encoded by the nucleotide sequence set forth in SEQ ID
NO:67 (corresponding to the amino acid sequence set forth in SEQ ID NO:117) and a recombinant CPR1 gene encoding (SEQ ID NO:77, SEQ ID NO:78) was deleted for S.

rebaudiana UGT85C2 polypeptide (SEQ ID NO:5/SEQ ID NO:6, SEQ ID NO:7). Sixteen independent clones were grown in Synthetic Complete (SC) medium at 30 C for 5 days with shaking (400 rpm for deep wells) prior to harvest. Culture samples (without cell removal) were heated in the presence of DMSO for detection of total glycoside levels with LC-MS.
[00206] As shown in Figure 4A, culture samples of cells deleted of UGT85C2 did not accumulate ent-kaurenol glycosides (ent-kaureno1+3GIc (isomer 1), ent-kaureno1+3GIc (#6), or ent-kaurenol_2GIc (#8), as compared to the control strain (not deleted for UGT85C2). This result suggests that UGT85C2 is responsible for the 19-0-glucosylation of ent-kaurenol. Also as shown in Figure 4A, culture samples of cells deleted of UGT85C2 did accumulate ent-kaurenoic acid glycosides (ent-kaurenoic acid+2GIc (#7), ent-kaurenoic acid+3GIc (isomer 1), and ent-kaurenoic acid+3GIc (isomer 2)). Whereas control samples accumulated 13-SMG, culture samples of cells deleted of UGT85C2 accumulated 19-SMG, steviol, stevio1+2GIc (#23), and stevio1+3GIc (#34). See Figures 4B and 4C. Stevio1+2GIc (#23) and stevio1+3GIc (#34) likely have two or three glucose moieties, respectively, attached on the 19 position of the steviol backbone.
[00207] Structures of isolated tri-glycosylated ent-kaurenoic acid, elucidated by NMR, are shown in Figure 7A, along with a structure of tri-glycosylated ent-kaurenol.
These structures were solved by means of standard homo- and heteronuclear multipulse NMR
experiments, 1H,1H-COSY, 1H,1H-ROESY, 1H,13C-HSQC, and 1H,13C-HMBC. Compounds were dissolved in 60 pL DMSO-d6 and measured at 25 C. Spectra of these compounds were acquired on an 800 MHz Bruker Avance instrument (800 MHz for 1H, 201 MHz for 13C) equipped with a cryogenic probe (5 mm CPTCI 1H-13C/15N/D Z-GRD Z44909/0010). In addition, 1H-NMR spectra were obtained for 3 molecules detected by LC-MS that were concordant with a general ent-kaurenoic acid+2G1c, ent-kaureno1+3GIc (isomer 2), and ent-kaurenol+Glc+GIcNAc structures. See Figures 8A-8L for 1H NMR spectra and 1H and 13C NMR chemical shifts for these compounds.
[00208] UGT85C2 variants were subsequently cloned into USER vectors (for integration at ChrXII-1) using a forward primer (SEQ ID NO:215) and a reverse primer (SEQ ID
NO:216) and the PGK1 promoter. The UGT85C2 variants were then integrated into the steviol glycoside-producing strain deleted of UGT85C2. Transformants were re-streaked from transformation plates. Pre-cultures were set up from re-streaked plates in 500 pL synthetic complete-URA
(SC-URA) media in a 96 deep well plate (DWP) and grown at 30 C and 300 rpm overnight.
Cultures were set up by transferring 50 pL of the pre-cultures to a 96 well DWP comprising 500 pL SC-URA media.
[00209] After 1 day of incubation, cultures were set up from pre-cultures (50 pL in 500 pL
SC-URA) and grown in Duetz system for 5 days (same conditions as for pre-cultures). The 0D600 was measured on plate reader in a 1:10 dilution, and samples were harvested by transferring 50 pL sample to 50 pL 100% DMSO. The mixtures were heated to 80 C
for 10 min and subsequently spun down (4000 rcf, 4 C, 10 min). 15 pL of each supernatant were mixed with 105 pL 50% DMSO (total dilution of 1:16), and the samples were analyzed by LC-MS.
Example 7: Assessment of UGT85C2 Variant Activity in Cell Lysates
[00210] Purified variant UGT85C2 DNA from Example 6 was individually transformed into XJB autolysis z-competent cells. Pre-cultures of three colonies from each transformation plate were inoculated into 600 pL LB comprising kanamycin (600 mg/L) and incubated overnight at 200 rpm and 37 C in a 96 well DWP. Protein production and cell wall degradation were induced by transferring 50 pL of the pre-cultures to a new 96 well DWP comprising 1mL/well of NZCYM
broth comprising kanamycin (600 mg/L) + 3 mL/L 1M Arabinose and 100 pL/L 1M
IPTG.
Cultures were incubated at 20 C, 200 rpm for approximately 20 h before pelleting the cells (4000 rcf, 5 min, 4 C) and removing the supernatant. To each well, 50 pL GT
buffer with protease inhibitor (cOmpleteTM, Mini, EDTA-free Protease Inhibitor Cocktail Tablets, 11836170001 Roche) was added. Pellets were resuspended by shaking at 200 rpm for 5 min at 4 C. A 75 pL aliquot of each sample was transferred to a PCR plate and frozen at -80 C. Pellets were thawed at room temperature, and 25 pL/well DNAse mix (2,39 mL 4x binding buffer + 50 pL DNAse I (1.4 mg/mL) + 60 pL MgC12 (1 M) per plate) were added when samples were nearly thawed. The plate was incubated at room temperature for 5 min with gentle shaking and subsequently centrifuged at 4000 rcf for 5 min. Each supernatant was transferred to a fresh PCR plate for activity measurements.
[00211] Each supernatant was incubated in an assay reaction mix comprising a final concentration of 100 mM Tris (pH 8.0), 4 mM MgC12, 1 mM KCI, 300 pM UDP-Glucose, and 100 pM substrate. The substrates were either steviol or 19-SMG. A purified wild-type UGT85C2 enzyme and a UGT85C2 bacterial lysate were used as positive controls.
Reactions were incubated at 30 C (on a plate shaker), and the reactions were stopped after 20 min, 40 min, and 19 h by mixing 20 pL sample with 20 pL 100% DMSO. The samples were further diluted by adding 60 pL 50% DMSO and subsequently analyzed by LC-MS. AUC values corresponding to measured 13-SMG, 19-SMG, rubusoside, and steviol levels are shown in Tables 8A-C.
Table 8A. Measured 13-SMG and steviol AUC values in UGT85C2 variant activity assay using steviol as a substrate.
13-SMG Steviol UGT85C2 Variant 20 min 40 min 19 h 20 min 40 min 19 h F48S (SEQ ID NO:150) 38195 55395 76045 21355 9955 F48H (SEQ ID NO:151) 49840 64105 79000 17670 4035 F48Y (SEQ ID NO:152) 36980 53005 83100 26675 16135 F48R (SEQ ID NO:153) 37990 55510 71810 25540 11075 F48Q (SEQ ID NO:154) 33660 46010 72550 30565 16135 F48W (SEQ ID NO:155) 37580 56220 76490 25280 8615 F48T (SEQ ID NO:156) 40505 57280 78080 20405 10340 149V (SEQ ID NO:157) 48345 60720 75420 17545 4305 S84G (SEQ ID NO:158) 33960 50770 76070 29500 15870 584A (SEQ ID NO:159) 43135 62000 75715 21445 5190 584C (SEQ ID NO:161) 25780 39330 71060 34285 22700 584V (SEQ ID NO:164) 27045 43200 74505 32100 17715 P86R (SEQ ID NO:165) 23240 34440 71955 33670 25395 P86G (SEQ ID NO:166) 28000 43525 74300 27640 14380 187H (SEQ ID NO:167) 7290 10465 43495 51340 187P (SEQ ID NO:168) 32165 48565 76700 29475 13945 187Y (SEQ ID NO:170) 36905 47250 71390 31220 14065 L91K (SEQ ID NO:171) 25810 37830 72435 29455 19015 L91R (SEQ ID NO:172) 27560 40235 75830 34275 22140 L92F (SEQ ID NO:174) 49205 62540 72385 15635 3570 Table 8B. Measured 13-SMG, 19-SMG, and rubusoside AUC values in UGT85C2 variant activity assay using 19-SMG as a substrate.
19-SMG rubusoside UGT85C2 Variant 20 min 40 min 19 h 20 min 40 min 19 h F48S (SEQ ID NO:150) 171625 147690 3720 18935 30650 F48H (SEQ ID NO:151) 165365 129495 1830 24415 40520 F48Y (SEQ ID NO:152) 161680 128705 2815 23130 39385 F48R (SEQ ID NO:153) 166035 142095 6120 17335 30075 F48Q (SEQ ID NO:154) 169560 145130 3235 16570 28495 F48W (SEQ ID 168175 147640 3920 16040 28030 95530 NO:155) F48T (SEQ ID NO:156) 166190 134425 2960 22445 37520 149V (SEQ ID NO:157) 170460 133705 1935 20340 35300 584G (SEQ ID 175515 147045 3165 14645 24745 91945 NO:158) 584A (SEQ ID NO:159) 163565 131735 1790 19805 31845 584C (SEQ ID NO:161) 183175 159805 44230 11040 17040 584V (SEQ ID NO:164) 183415 168240 6600 11975 20075 P86R (SEQ ID NO:165) 186925 154290 12670 12075 20350 P86G (SEQ ID 175265 146080 5720 17660 29815 93195 NO:166) 187H (SEQ ID NO:167) 197170 191250 149025 3045 5300 187P (SEQ ID NO:168) 167935 143945 8795 16675 28290 187Y (SEQ ID NO:170) 176815 142820 4750 16635 26615 L91K (SEQ ID NO:171) 188110 182210 177120 5350 8545 L91R (SEQ ID NO:172) 188750 180040 149165 7535 12140 L92F (SEQ ID NO:174) 187295 155170 2695 11335 22340 Table 8C. Measured 13-SMG, 19-SMG, rubusoside, and steviol AUC values in control UGT85C2 assays.
13-SMG 19-SMG rubusoside Steviol 20 min 40 19h 20 min 40 min 19h 20 40 min 19h 20 40 19h min min min min Substrate: Steviol 60635 67575 73750 490 (SEQ ID NO:7) Substrate: 19-SMG 53380 4635 1775 85560 108620 (SEQ ID NO:7) Substrate: Steviol 53745 46585 No UGT85C2 Substrate: 19-SMG 224605 206230 199490 No UGT85C2
[00212] Accumulation of 19-SMG and rubusoside was not observed in UGT85C2 variant activity assays using steviol as a substrate. Using steviol as the substrate, the F48H, F48Y, F48T, I49V, S84A, and L92F UGT85C2 variants demonstrated high activity during incubation periods of under 40 min, and the F48H, F48Y, F48T, and I49V UGT85C2 variants demonstrated high activity during incubation periods of over 40 min (Table 8A). Using 19-SMG as the substrate, the F48H, F48Y, F48T, I49V, and S84A UGT85C2 variants demonstrated high activity during incubation periods of under 40 min, and the F48H, I49V, S84A, S84V, L91K, and L92F UGT85C2 variants, as well as the wild-type UGT85C2, demonstrated high activity during incubation periods of over 40 min (Table 8B). Slow conversion of steviol and 19-SMG was observed for UGT85C2 I87H (Tables 8A and 8B).
[00213] 13-SMG/rubusoside ratios were calculated for the UGT85C2 variants.
A high 13-SMG/rubusoside ratio indicates preference of a UGT85C2 variant for steviol, whereas a low 13-SMG/rubusoside ratio indicates preference of a UGT85C2 variant for 19-SMG. The L91K, L91R, and L92F UGT85C2 variants demonstrated a high 13-SMG/rubusoside ratio, whereas the F48Y, F48T, P86G UGT85C2 variants demonstrated a low 13-SMG/rubusoside ratio.
[00214] The UGT85C2 variants were found to convert steviol to rubusoside after 24 h.
Rubusoside levels (in AUC) are shown in Figure 5. Mutations in the amino acid 48 and 49 positions produced increased levels of rubusoside, as compared to the control.
The variants with mutations in amino acids at position 86, 91 and 92 seem to produce lower levels of rubusoside.
Example 8: Evaluation of UGT76G1 Variants
[00215] UGT76G1 variants were tested in a modified version of a steviol glycoside-producing S. cerevisiae strain as described in Example 2 to determine the effects on steviol glycosides, tri-glycosylated ent-kaurenol, and tri-glycosylated ent-kaurenoic acid levels. The background strain was described in Example 9 of WO 2014/122227, wherein both copies of UGT76G1 were deleted by homologous recombination using selective markers. The strain comprised a reintegrated wild-type UGT76G1 (WT control) or variants of UGT76G1 at the chromosome level.
[00216] Expression of UGT76G1 H155L (SEQ ID NO:184) increased the ratio of RebM/RebD
produced, as compared to wild-type UGT76G1. Expression of UGT76G1 Q23H (SEQ ID

NO:181), UGT76G1 T146G (SEQ ID NO:183), UGT76G1 L257G (SEQ ID NO:185), or UGT76G1 5283N (SEQ ID NO:188) in the strain all resulted in increased accumulation of ent-kaurenoic acid+2GIc (#7), 1,2-bioside, 1,2-stevioside, RebE, RebD, stevio1+5GIc (#22), and stevio1+6GIc (isomer 1), increased the ratio of RebD/RebM produced, and decreased accumulation of RebB and RebA, as compared to wild-type UGT76G1. See Tables 9A-9C.
Specifically, expression of UGT76G1 T146G (SEQ ID NO:183), resulted in increased accumulation of ent-kaurenoic acid+3GIc (isomer 1), stevio1+3GIc (#1), and Stev3GIc (#34), as compared to wild-type UGT76G1. Expression of UGT76G1 L257G (SEQ ID NO:185) increased the amount of stevio1+7GIc (isomer 2), as compared to wild-type UGT76G1.
Expression of UGT76G1 5283N (SEQ ID NO:188) increased the amount of stevio1+3GIc (#1) and Stev3GIc (#34), as compared to wild-type UGT76G1. See Tables 9A-9C.
Table 9A. Accumulation of steviol glycosides (in pM) in a host comprising wild-type UGT76G1 or a UGT76G1 variant.
13-SMG 1,2- RebB RebA RebE RebD RebM
bioside Wild-type (SEQ ID 13.5 29.3 NO:9) 3.8 N/A 1.5 0.4 4.7 1.9 N/A 5.2 2.5 15.5 H155L (SEQ 13.9 38.8 ID NO:184) 2.4 N/A 1.8 0.2 6.5 1.5 N/A 2.1 0.3 12.6 Q23H (SEQ ID 13.4 17.7 +
- 1.9 0.7 NO:181) 2.2 1.8 0.4 0.9 0.1 1.3 0.2 4.6 0.6 6.4 T146G (SEQ 13.9 14.1 +
- 1.1 0.2 ID NO:183) 2.7 2.0 0.4 0.6 0.3 0.7 0.5 7.4 1.9 3.5 L257G (SEQ 13.6 32.0 7.0 1.5 ID NO:185) 0.9 1.2 0.1 0.9 0.2 2.3 0.3 2.8 0.4 6.1 S283N (SEQ 13.5 + 14.4 +
- 0.9 0.4 ID NO:188) 1.4 2.1 0.4 0.5 0.1 0.3 0.5 7.9 1.0 3.9 Q23H+H155L
(SEQ ID 12.4 22.4 +
- 8.4 3.4 NO:217) 1.1 1.4 0.3 0.8 0.1 1.9 0.5 4.0 0.4 5.9 T146G+H155L
(SEQ ID 13.8 26.5 +
- 9.5 1.9 NO:218) 1.3 1.4 0.2 0.8 0.1 2.2 0.1 3.4 0.4 2.5 L257G+H155L
(SEQ ID 14.1 23.8 1.5 NO:219) 1.3 0.9 0.4 1.0 0.1 3.1 0.5 1.8 0.5 5.2 S283N+H155L
(SEQ ID 13.4 10.1 +
- 1.2 0.6 NO:220) 2.6 2.3 0.5 0.5 0.3 0.3 0.5 7.2 1.8 4.3 Table 9B. Accumulation of steviol glycosides, glycosylated ent-kaurenoic acid, or glycosylated kaurenol (in AUC) in a host comprising wild-type UGT76G1 or a variant.

KA+2Gic KA+3Gic KA+3Gic KL+2Gic KL+3Gic 1,2- stevio1+3Gic (#7) (isomer (isomer (#8) (isomer stevioside (#1) 1) 2) land isomer 2) Wild-type N/A N/A
(SEQ ID 859 N/A
NO:9) N/A N/A 1089 887 668 H155L (SEQ 1862 N/A 550 N/A
ID NO:184) N/A N/A 1825 1035 874 754 Q23H (SEQ ID 3118 592 N/A N/A 6716 NO:181) 1068 1165 N/A 966 T146G (SEQ 3109 1355 N/A N/A 8313 ID NO:183) 1441 951 N/A 1498 L257G (SEQ 2562 1062 N/A N/A 5716 N/A
ID NO:185) 1267 1199 N/A 837 S283N (SEQ 3872 1200 N/A N/A 8572 +

ID NO:188) 1086 1929 N/A
Q23H+H155L N/A N/A
(SEQ ID 2690 236 6690 +

NO:217) 423 N/A 668 734 T146G+H155L N/A N/A
(SEQ ID 2416 6172 +

NO:218) 555 N/A N/A 524 L257G+H155L N/A 222 (SEQ ID 1634 212 1524 628 5458 +

NO:219) 1227 600 1318 S283N+H155L 408 N/A
(SEQ ID 3886 496 1154 8036 +

NO:220) 750 929 N/A 1601 -KA:ent-kaurenoic acid -KL:ent-kaurenol Table 9C. Accumulation of steviol glycosides (in AUC) in a host comprising wild-type UGT76G1 or a UGT76G1 variant.
steviol stevio1+4 stevio1+4 stevio1+5 stevio1+5 steviol stevio1+7 +3 Glc Glc (#26) Glc (#33) Glc (#22) Glc (#25) +6 Glc Glc (#34) (isome (isomer r 1) 2) Wild-type N/A
(SEQ ID 2443 N/A
NO:9) N/A 1164 N/A N/A N/A

(SEQ ID 1020 1039 N/A
NO:184) N/A 731 N/A N/A N/A
Q23H 472 19804 N/A 7350 +

(SEQ ID 507 818 726 N/A 4600 NO:181) (SEQ ID 1262 1509 38469 7365 +

NO:183) 605 376 114 302 8953 (SEQ ID 104 1038 11638 10722 3870 NO:185) 294 459 N/A 2268 1871 2463 (SEQ ID 1168 1572 44460 12174 NO:188) 655 625 104 294 11455 5214 N/A
Q23H+H15 N/A

5L (SEQ ID 122 16600 4404 +

NO:217) 345 964 459 N/A 3617 2744 -T146G+H1 N/A

55L (SEQ 212 1114 14362 2498 +

ID NO:218) 383 192 N/A 1802 2743 -L257G+H1 N/A

(SEQ ID 6354 2408 + 5780 NO:219) N/A 782 725 N/A 4578 2584 -S283N+H1 N/A
55L (SEQ 1186 1020 38410 3864 +

ID NO:220) 673 739 N/A 17463
[00217] The double UGT76G1 variants were also tested. The double variants were:
UGT76G1 Q23H H155L (SEQ ID NO:217), UGT76G1 T146G H155L (SEQ ID NO:218), UGT76G1 L257G H155L (SEQ ID NO:219), and UGT76G1 5283N H155L (SEQ ID NO:220).
Double variants UGT76G1 Q23H H155L (SEQ ID NO:217), UGT76G1 T146G H155L (SEQ
ID
NO:218), and UGT76G1 L257G H155L (SEQ ID NO:219) resulted in increased RebM
accumulation, as compared to the three single variants UGT76G1 Q23H (SEQ ID
NO:181), UGT76G1 T146G (SEQ ID NO:183), and UGT76G1 L257G (SEQ ID NO:185). See Tables 90. Specifically, expression of UGT76G1 Q23H H155L (SEQ ID NO:217) increased the amount of RebM and stevio1+7GIc (isomer 2), compared to the UGT76G1 Q23H (SEQ ID
NO:181) variant. Expression of UGT76G1 T146G H155L (SEQ ID NO:218) increased accumulation of RebA, RebD, RebM, and stevio1+7GIc (isomer 2) and decreased accumulation of ent-kaurenoic acid+3GIc (isomer1), 1,2-bioside, 1,2-stevioside, stevio1+3GIc (#1), Stev3GIc (#34), RebE, and stevio1+5GIc (#22), as compared to the UGT76G1 T146G (SEQ ID NO:183) variant.
Expression of UGT76G1 L257G H155L (SEQ ID NO:219) increased accumulation of ent-kaurenoic acid+3GIc (isomer 2), RebA, and RebM and decreased accumulation of RebE and stevio1+6GIc (isomer 1), as compared to the UGT76G1 L257G (SEQ ID NO:185) variant. See Tables 9A-90. Thus, synergistic effects were observed for UGT76G1 double variants.
[00218] UGT76G1 variants were also analyzed in a modified version of the strain described above, which comprised a higher copy number of UGT91D2e (SEQ ID NO:10, SEQ ID
NO:11), UGT74G1 (SEQ ID NO:3, SEQ ID NO:4), and ATR2 (SEQ ID NO:91, SEQ ID NO:92).
Steviol glycoside-producing S. cerevisiae strains expressing UGT76G1 variants that resulted in increased RebD levels, including UGT76G1 Q23H, UGT76G T146G, and 5283N, also increased accumulation of ent-kaurenoic acid+2GIc (#7) and ent-kaurenoic acid+2GIc (isomer 1) but decreased accumulation of ent-kaurenoic acid+3GIc (isomer 2), compared to steviol glycoside-producing S. cerevisiae strains expressing wild-type UGT76G1. See Figure 9A.
UGT76G1 variants that increased RebD levels also increased accumulation of ent-kaureno1+2G1c (#8) but decreased accumulation of ent-kaureno1+3GIc (isomer 1) co-eluted with ent-kaureno1+3GIc (#6) (Figure 9B).
[00219] Expression of the UGT76G1 H155L variant (SEQ ID NO:184), a variant that increased levels of RebM, resulted in decreased accumulation of ent-kaurenoic acid+2GIc (#7) and ent-kaurenoic acid+3GIc (isomer 1) (Figure 9A). Levels of ent-kaurenol glycosides were not significantly altered upon expression of UGT76G1 variants that increased levels of RebM, compared to strains expressing wild-type UGT76G1 (Figure 9B).
[00220] Levels of 13-SMG, 1,2-bioside, rubusoside, RebA, RebB, RebD, RebE, RebM, RebG
(1,3-stevioside), stevio1+3GIc (#1), stevio1+4GIc (#26), stevio1+5GIc (#22), stevio1+5GIc (#24), stevio1+5GIc (#25), stevio1+6GIc (isomer 1), and stevio1+6GIc (#23) produced in the steviol glycoside-producing strain are shown in Figures 10A-10C. Expression of UGT
variants that resulted in increased RebD levels also increased accumulation of stevio1+5GIc (#22), 1,2-stevioside, stevio1+6GIc (isomer 1), and Stevio+3GIc (#1) but decreased accumulation of stevio1+4GIc (#26), stevio1+5GIc (#24), and RebG (1,3-stevioside) (Figure 10A). Expression of UGT76G1 H155L (SEQ ID NO:184) resulted in increased accumulation of stevio1+5GIc (#25) but decreased accumulation of 1,2-stevioside, stevio1+3GIc (#1), stevio1+4GIc (#26), stevio1+5GIc (#22), stevio1+6GIc (isomer 1), and stevio1+6GIc (#23) (Figure 10B). Expression of UGT76G1 S253W (SEQ ID NO:186) resulted in decreased accumulation of 1,2-stevioside and stevio1+6GIc (isomer 1) (Figure 10B). Expression of UGT76G1 284G resulted in increased accumulation of 1,2-stevioside and stevio1+6GIc (isomer 1) but decreased accumulation of RebG, stevio1+4GIc (#26), stevio1+5GIc (#25), and stevio1+6GIc (#23) (Figure 10B). Figure 100 shows accumulation of 13-SMG, 1,2-bioside, rubusoside, RebA, RebB, RebD, RebE, and RebM
in S. cerevisiae expressing wild-type UGT76G1 (SEQ ID NO:9) or a UGT76G1 variant that increases accumulation of RebD or RebM.
[00221] The steviol glycoside-producing strain comprising a higher copy number of UGT91D2e (SEQ ID NO:10, SEQ ID NO:11), UGT74G1 (SEQ ID NO:3, SEQ ID NO:4), and ATR2 (SEQ ID NO:91, SEQ ID NO:92) was further tested in a separate experiment.
As shown in Tables 9D-9F, expression of UGT76G1 H155L (SEQ ID NO:184) resulted in increased accumulation of stevio1+5GIc (#25), increased the ratio of RebM/RebD produced, and decreased accumulation of 1,2-bioside, stevio1+3GIc (#1), RebE, stevio1+6GIc (isomer 1), and stevio1+6GIc (#23), as compared to wild-type UGT76G1. Expression of UGT76G1 Q23H (SEQ
ID NO:181), UGT76G1 T146G (SEQ ID NO:183), UGT76G1 L257G (SEQ ID NO:185), or UGT76G1 5283N (SEQ ID NO:188) increased accumulation of 1,2-bioside, 1,2-stevioside, stevio1+3GIc (#1), Stev+3GIc (#34), RebE, and stevio1+5GIc (#22), increased the ratio of RebD/RebM produced, and decreased accumulation of RebG, RebA, stevio1+5GIc (#25), stevio1+7GIc (isomer 2), and stevio1+7GIc (isomer 5). Specifically, expression of UGT76G1 Q23H (SEQ ID NO:181) resulted in increased accumulation of rubusoside, stevio1+6GIc (isomer 1) and decreased accumulation of RebB and stevio1+5GIc (#24). Expression of T146G (SEQ ID NO:183) resulted in increased accumulation of rubusoside and decreased accumulation of RebB, stevio1+5GIc (#24) and stevio1+6GIc (#23). Expression of L257G (SEQ ID NO:185) resulted in increased accumulation of stevio1+6GIc (isomer 1).
Expression of UGT76G1 5283N (SEQ ID NO:188) resulted in increased accumulation of rubusoside and decreased accumulation of RebB, stevio1+5GIc (#24) and stevio1+6GIc (#23).
See Tables 9D-F.
Table 9D.
Accumulation of steviol glycosides (in pM) in a host comprising wild-type UGT76G1 or a UGT76G1 variant.
13- 1,2- Rubu RebG RebB RebA RebE RebD RebM
SMG bioside Wild-type 430 +
(SEQ ID 37.6 1.3 1.2 0.2 8.4 32.5 0.4 30.4 + = -NO:9) 8.8 0.5 0.2 0.2 2.3 7.5 0.1 12.5 - 9'6 64.5 H155L (SEQ 35.3 0.4 1.3 0.2 8.9 35.2 0.1 5.7 7.1 ID NO:184) 7.0 0.1 0.1 0.2 2.1 9.3 0.1 1.8 1 0 +
Q23H (SEQ ID 40.8 11.1 2.4 4.3 7.2 11.8 35.1 + =
-0- .4 NO:181) 6.9 1.5 0.4 N/A 1.3 2.0 4.5 6.5 0 2 +
T146G (SEQ 41.4 16.1 3.1 1.5 2.4 19.2 15.0 + =
0- .2 ID NO:183) 6.9 1.4 0.4 N/A 0.5 1.1 3.2 5.3 2 3 +
L257G (SEQ 32.4 6.9 1.8 5.2 12.1 4.7 41.7 + =
-ID NO:185) 6.2 1.0 0.5 N/A 1.8 4.8 1.6 10.4 ' 9 0 3 +
S283N (SEQ 39.8 15.1 2.6 1.5 2.9 16.2 19.2 + =
-- 0.1 ID NO:188) 7.2 2.8 0.4 N/A 0.5 1.2 4.8 6.9 Q23H+H155L
3 0 +
(SEQ ID 39.4 9.0 2.1 4.7 8.3 8.8 34.1 +
= -- 1.2 NO:217) 4.5 1.3 0.2 N/A 0.9 2.6 1.6 4.5 T146G+H155L
3 1 +
(SEQ ID 33.0 8.5 1.9 3.8 9.2 6.6 36.5 +
= -- 0.9 NO:218) 8.0 2.0 0.7 N/A 1.0 2.9 1.7 4.7 L257G+H155L

(SEQ ID 44.4 4.9 1.5 8.2 19.2 3.4 47.8 + =
+ -- 3.3 NO:219) 6.6 0.9 0.3 N/A 1.2 4.0 1.0 4.5 S283N+H155L
0 7 +
(SEQ ID 42.9 14.5 2.8 2.1 2.7 16.7 17.2 + =
-- 0.3 NO:220) 6.6 1.1 0.2 N/A 0.7 0.9 1.9 3.7 Table 9E.
Accumulation of steviol glycosides, glycosylated ent-kaurenoic acid, or glycosylated kaurenol (in AUC) in a host comprising wild-type UGT76G1 or a variant.
KA+2 KA+3 KA+3 KL+2 KL+3 19- 1,3-1,2- stevio stevio Glc Glc Glc Glc Glc SMG
biosid stevio 1+3Gic 1+3Gic (#7) (isom (isom (#8) (isom e side (#1) (#34) en) er 2) en 1 and isome r 2) Wild-type 47650 12328 274 23410 1512+
(SEQ ID 14444 2472 8102 8 2174 775 + 2226 +

NO:9) 5537 1360 20783 4937 20872 1054 10331 1961 (SEQ ID 1096 + 1770 4 2072 13466 N/A
NO:184) 1570 N/A 17847 1118 33369 940 (SEQ ID 2 10386 2914 4 + 2364 0 NO:181) 26599 2233 2162 22523 11295 520 50824 6924 (SEQ ID 5 7339 9 21515 1961 5 NO:183) 18966 2016 N/A 21693 3812 1049 38885 6704 (SEQ ID 2 9732 7486 4 + 2010 6 13040 +2086 NO:185) 39204 3604 3428 34855 22294 +
(SEQ ID 0 8722 2 19864 1980 8 +

NO:188) 55275 3756 N/A 63472 6586 875 58796 10712 Q23H+H1 N/A
55L (SEQ 10793 86190 16226 +3184 NO:217) 18511 944 3586 13792 7629 674 12368 3180 -T146G+H 10414 9346 13674 98980 81762 2034 N/A 13851 18846 155L 6 1964 4859 + 768 0 (SEQ ID 17815 30306 19834 32208 NO:218) L257G+H N/A

(SEQ ID 7974 34450 34730 99436 2800 0 NO:219) 17561 1665 6021 9050 S283N+H N/A

(SEQ ID 4 8168 1706 4 31296 2694 6 25406 +6048 NO:220) 15045 1243 1880 25165 6636 574 11694 6048 --KA:ent-kaurenoic acid -KL:ent-kaurenol Table 9F.
Accumulation of steviol glycosides (in AUC) in a host comprising wild-type UGT76G1 or a UGT76G1 variant.
steviol+ steviol+ steviol+ steviol+ steviol+ steviol+ steviol+ steviol+ Ste 4GIc 5GIc 5GIc 5GIc 6GIc 6GIc 7GIc 7GIc viol (#26) (#22) (#24) (#25) (isomer (#23) (isomer (isomer 1) 2) 5) Wild- N/A
type (SEQ ID 38936 3288 2194 9068 12294 5838 13784 7630 NO:9) 21188 3892 2020 3994 10105 2979 4806 3054 (SEQ ID 20000 178 1530 29526 122 2000 6494 10782 NO:184) 4629 503 2310 15999 345 830 2530 2519 (SEQ ID 26366 161044 26590 3108 2964 918 NO:181) 7357 57250 N/A 3671 1514 1547 1268 (SEQ ID 25070 224315 10320 304 322 286 NO:183) 6192 53331 N/A 3647 804 853 756 L257G 6 +
(SEQ ID 17638 81252 258 31616 5088 5154 1590 +

NO:185) 5814 31941 730 5164 1171 1398 1335 (SEQ ID 24980 219964 19666 846 264 296 NO:188) 8098 61935 N/A 5418 1170 747 837 Q23H+H N/A N/A

(SEQ ID 23100 142460 15108 3582 5996 596 NO:217) 2234 24407 N/A 1958 819 1705 T146G+ N/A N/A

(SEQ ID 19064 120990 13048 4288 4640 1306 NO:218) 3666 34224 N/A 2270 889 1866 L257G+ N/A N/A

(SEQ ID 17126 56416 928 17756 5856 15114 2230 NO:219) 2237 15937 1293 2361 960 1900 985 S283N+ N/A N/A

(SEQ ID 23536 213846 11222 1162 1042 NO:220) 2818 31505 N/A 2649 1288 1117 N/A
[00222] Expression of UGT76G1 Q23H H155L (SEQ ID NO:217) increased accumulation of ent-kaurenoic acid+3GIc (isomer 2) and ent-kaureno1+3GIc (isomer 1) and decreased accumulation of ent-kaureno1+2GIc (#8) and stevio1+6GIc (isomer 1), as compared to UGT76G1 Q23H (SEQ ID NO:181). UGT76G1 T146G H155L (SEQ ID NO:218) increased accumulation of ent-kaurenoic acid+3GIc (isomer 2), ent-kaureno1+3GIc (isomer 1), RebB, RebA, RebD, stevio1+6GIc (#23), and stevio1+7GIc (isomer 2) and decreased accumulation of ent-kaurenoic acid+2GIc (#7), ent-kaureno1+2GIc (#8), 1,2-bioside, rubusoside, 1,2-stevioside, RebE, stevio1+5GIc (#22), as compared to UGT76G1 T146G (SEQ ID NO:183). Expression of UGT76G1 L257G H155L (SEQ ID NO:219) increased accumulation of ent-kaurenoic acid+3GIc (isomer 2), ent-kaureno1+3GIc (isomer 1), and stevio1+7GIc (isomer 2) and decreased accumulation of ent-kaureno1+2GIc (#8), 1,2-bioside, and stevio1+6GIc (isomer 1), as compared to UGT76G1 L257G (SEQ ID NO:185). As well, UGT76G1 L257G H155L (SEQ ID NO:219) increased accumulation of RebD, as compared to wild-type UGT76G1. Expression of UGT76G1 5283N H155L (SEQ ID NO:220) decreased accumulation of stevio1+6GIc (isomer 1), as compared to UGT76G1 5283N (SEQ ID NO:188). See Tables 9D-F.
[00223]
UGT76G1 variants were also expressed in a steviol glycoside-producing strain comprising an extra copy of CPR1 (SEQ ID NO:77, SEQ ID NO:78), an extra copy of SrKAHe1 (SEQ ID NO:93, SEQ ID NO:94), and an extra copy of a UGT76G1 (SEQ ID NO:8, SEQ
ID
NO:9) or a UGT76G1 variant. Accumulation of steviol glycosides, tri-glycosylated ent-kaurenol, and tri-glycosylated ent-kaurenoic acid levels were measured. See Figure 11.
[00224] UGT76G1 variants that increased accumulation of RebD or RebM were also expressed in a steviol glycoside production S. cerevisiae strain comprising an extra copy of CPR1 (SEQ ID NO:77, SEQ ID NO:78) and an extra copy of SrKAHe1 (SEQ ID NO:93, SEQ ID
NO:94). The control steviol glycoside production strain comprised three copies of wild-type UGT76G1 (SEQ ID NO:9), and the variant-comprising strains comprised two copies of wild-type UGT76G1 (SEQ ID NO:9) and one copy of a UGT76G1 variant. Figure 11A shows levels of ent-kaurenoic acid+2GIc (#7), ent-kaurenoic acid+3GIc (isomer 1), ent-kaurenoic acid+3GIc (isomer 2), ent-kaureno1+2GIc (#8), and ent-kaureno1+3GIc (isomer 1) co-eluted with ent-kaureno1+3G1c (#6) in production strains expressing wild-type UGT76G1 (SEQ ID
NO:9), UGT76G1 Q23H (SEQ ID NO:181), UGT76G1 I26W (SEQ ID NO:182), UGT76G1 T146G (SEQ

ID NO:183), UGT76G1 H155L (SEQ ID NO:184), UGT76G1 L257G (SEQ ID NO:185), or UGT76G1 5283N (SEQ ID NO:188). Total levels of glycosylated ent-kaurenoic acid (ent-kaurenoic acid+2GIc (#7) + ent-kaurenoic acid+3GIc (isomer 1) + ent-kaurenoic acid+3GIc (isomer 2)) were most significantly increased in production strains expressing (SEQ ID NO:181), UGT76G1 I26W (SEQ ID NO:182), and UGT76G1 L257G (SEQ ID
NO:185) (Figure 11B), and total levels of glycosylated ent-kaurenol (ent-kaureno1+3GIc (isomer 1) co-eluted with ent-kaureno1+3GIc (#6) and ent-kaureno1+2GIc (#8) were most significantly affected for production strains expressing UGT76G1 Q23H (SEQ ID NO:181), UGT76G1 I26W
(SEQ ID
NO:182), and UGT76G1 T146G (SEQ ID NO:183) (Figure 110).
[00225] Figure 11D and 11E show accumulation of 1,2-bioside, 1,2-stevioside, stevio1+3GIc (#1), stevio1+4GIc (#26), stevio1+5GIc (#22), stevio1+5GIc (#24), stevio1+5GIc (#25), stevio1+6GIc (isomer 1), stevio1+6GIc (#23), stevio1+7GIc (isomer 2), stevio1+7GIc (isomer 5), 13-SMG, rubusoside, RebG (1,3-stevioside), RebA, RebB, RebD, RebE, and RebM in production strains expressing wild-type UGT76G1 (SEQ ID NO:9), UGT76G1 Q23H (SEQ ID NO:181), I26W (SEQ ID NO:182), UGT76G1 T146G (SEQ ID NO:183), UGT76G1 H155L (SEQ ID
NO:184), UGT76G1 L257G (SEQ ID NO:185), or UGT76G1 5283N (SEQ ID NO:188).
[00226] All UGT76G1 variants tested in Figure 11D showed decreased accumulation of stevio1+4GIc (#26). Expression of UGT76G1 Q23H (SEQ ID NO:181), UGT76G1 I26W
(SEQ ID
NO:182), UGT76G1 T146G (SEQ ID NO:183), UGT76G1 L257G (SEQ ID NO:185), or UGT76G1 5283N (SEQ ID NO:188), all of which increased production of RebD, resulted in decreased accumulation of stevio1+5GIc (#25), compared to a control strain expressing wild-type UGT76G1 (Figure 11D). However, expression of the UGT76G1 H155L (SEQ ID
NO:184) variant, which increased RebM production, resulted in increased accumulation of stevio1+5GIc (#25) (Figure 11D).
[00227] Expression of UGT76G1 Q23H (SEQ ID NO:181), UGT76G1 I26W (SEQ ID
NO:182), UGT76G1 T146G (SEQ ID NO:183), UGT76G1 L257G (SEQ ID NO:185), or UGT76G1 5283N (SEQ ID NO:188) resulted in increased accumulation of stevio1+6GIc (#23), compared to a control strain expressing wild-type UGT76G1, whereas expression of the UGT76G1 H155L (SEQ ID NO:184) variant resulted in decreased accumulation of stevio1+6GIc (#23) (Figure 11D). Expression of UGT76G1 Q23H (SEQ ID NO:181), UGT76G1 126W
(SEQ
ID NO:182), UGT76G1 T146G (SEQ ID NO:183), UGT76G1 L257G (SEQ ID NO:185), or UGT76G1 5283N (SEQ ID NO:188) resulted in increased accumulation of stevio1+7GIc (isomer 2), compared to a control strain expressing wild-type UGT76G1, whereas expression of the UGT76G1 H155L (SEQ ID NO:184) variant resulted in decreased accumulation of stevio1+7GIc (isomer 2) (Figure 11D). Expression of UGT76G1 Q23H (SEQ ID NO:181), UGT76G1 (SEQ ID NO:182), UGT76G1 T146G (SEQ ID NO:183), UGT76G1 L257G (SEQ ID NO:185), or UGT76G1 5283N (SEQ ID NO:188) resulted in increased accumulation of stevio1+7GIc (isomer 5) (Figure 11D).
[00228] The steviol glycoside-producing strain comprising a higher copy number of CPR1 (SEQ ID NO:77, SEQ ID NO:78) and SrKAHe1 (SEQ ID NO:93, SEQ ID NO:94) was further tested in a separate experiment. As shown in Tables 9G-9I, expression of (SEQ ID NO:184) reduced the levels of ent-kaurenoic acid+3GIc (isomer 1), RebD, stevio1+6GIc (#23), stevio1+7GIc (isomer 2), as compared to wild-type UGT76G1. Expression of UGT76G1 Q23H (SEQ ID NO:181), UGT76G1 T146G (SEQ ID NO:183), UGT76G1 L257G (SEQ ID
NO:185), or UGT76G1 5283N (SEQ ID NO:188) each reduced accumulation of stevio1+4GIc (#26) and stevio1+5GIc (#24), as compared to wild-type UGT76G1. Specifically, expression UGT76G1 T146G (SEQ ID NO:183) increased the amount of ent-kaurenoic acid+2GIc (#7), ent-kaurenoic acid+3GIc (isomer 1), RebD, stevio1+6GIc (#23), and stevio1+7GIc (isomer 2) and reduced the amount of RebG, stevio1+5GIc #25, as compared to wild-type UGT76G1.
Expression of UGT76G1 L257G (SEQ ID NO:185) increased accumulation of ent-kaurenoic acid+3GIc (isomer 1) and reduced accumulation of ent-kaurenoic acid+3GIc (isomer 2) and stevio1+5GIc (#25), as compared to wild-type UGT76G1. Expression of UGT76G1 5283N (SEQ
ID NO:188) increased accumulation of ent-kaurenoic acid+3GIc (isomer 1), RebD, stevio1+6GIc (isomer 1), and stevio1+7GIc (isomer 2) and reduced accumulation of RebG and stevio1+5G1 (#25), as compared to wild-type UGT76G1. Expression of UGT76G1 L257G H155L
reduced accumulation of ent-kaurenoic acid+3GIc (isomer 1), as compared to the single variant UGT76G1 L257G. Expression of the double variant UGT76G1 Q23H H155L reduced accumulation of stevio1+5GIc (#25), as compared to wild-type UGT76G1.
Expression of the double variant UGT76G1 5283N H155L reduced accumulation of ent-kaurenoic acid+3GIc (isomer 2), as compared to wild-type UGT76G1. See Tables 9G-91.

Table 9G.
Accumulation of steviol glycosides (in pM) in a host comprising wild-type UGT76G1 or a UGT76G1 variant.
13- 1,2- Rubu RebG RebB RebA RebE RebD RebM
SMG bioside Wild-type (SEQ ID 66.9 1.2 0.7 5.6 30.3 0.5 31' 0 +
199.3 - +

NO:9) 4.7 0.4 0.1 0.2 0.3 0.4 2.4 0.4 6.7 - 14.2 H155L (SEQ 63.1 + 1.3 0.9 5.5 29.6 0.1 12.0 + 210.0 -ID NO:184) 4.6 0.3 0.1 0.3 0.3 0.5 1.9 0.2 10.8 -179.2 Q23H (SEQ ID 62.2 + 0.8 0.2 5.2 27.7 0.6 42.0 +
- +19 6 NO:181) 13.9 0.4 0.1 0.3 0.3 0.9 3.3 0.2 9.8 -' 180.4 T146G (SEQ 64.8 1.0 0.1 5.3 27.9 0.8 46.2 +
- + 24 2 ID NO:183) 5.2 0.5 0.2 0.1 0.2 0.8 3.1 0.1 6.7 -' L257G (SEQ 68.7 0.6 0.2 5.5 29.6 0.6 45.6 6 + 187.3 +

ID NO:185) 9.2 0.4 0.1 0.4 0.3 0.6 3.4 0.4 9.3 -14.7 189.2 S283N (SEQ 67.4 + 0.7 0.1 5.7 32.0 0.8 52.7 +
-ID NO:188) 13.3 0.4 0.1 0.5 0.2 0.7 4.2 0.4 7.4 -Q23H+H155L
(SEQ ID 65.2 0.8 0.3 5.3 27.1 0.7 37.5 5 + 187.5 + 10 8 NO:217) 4.3 0.3 0.0 0.4 0.3 0.3 2.8 0.3 5.4 - ' T146G+H155L
(SEQ ID 64.3 0.8 0.1 5.4 27.3 0.7 40.0 + 171.2 -29.8 NO:218) 9.8 0.5 0.1 0.3 0.2 0.6 4.3 0.4 8.7 L257G+H155L
(SEQ ID 58.5 0.5 0.3 5.2 25.1 0.7 30.4 + 167.6 33.6 NO:219) 15.9 0.3 0.1 0.5 0.3 1.5 7.9 0.3 13.3 -S283N+H155L
(SEQ ID 61.2 0.6 0.0 5.2 25.0 0.6 37.5 NO:220) 11.8 0.4 0.1 0.5 0.0 1.0 5.5 0.5 12.0 - 35.2 Table 9H.
Accumulation of steviol glycosides, glycosylated ent-kaurenoic acid, or glycosylated kaurenol (in AUC) in a host comprising wild-type UGT76G1 or a variant.
KA+2GI KA+3GI KA+3GI KL+3GI 19-SMG 1,2- Rebl steviol+
c (#7) c c c steviosi 4GIc (isomer (isomer (isomer de (#26) 1) 2) 1 and isomer 2) Wild-type (SEQ ID 2422 1962 40290 11500 422 4712 NO:9) 419 383 3139 1169 270 656 N/A
H155L (SEQ 2894 418 40350 10326 376 4466 512 9086 ID NO:184) 401 841 2392 759 316 359 992 1374 Q23H (SEQ 3340 3044 41140 11404 476 4452 ID NO:181) 1018 747 5158 1306 317 595 N/A 771 T146G (SEQ 3362 + 2934 40636 10880 400 4600 ID NO:183) 509 399 5193 872 350 511 N/A
L257G (SEQ 2816 + 2712 34402 10820 254 4770 ID NO:185) 240 264 2377 708 272 642 N/A 674 S283N (SEQ 3114 2914 35830 11430 188 4986 5734 442 ID NO:188) 585 346 2929 641 348 562 N/A
Q23H+H155 L (SEQ ID 2622 2250 37176 10376 264 4404 NO:217) 286 408 3860 1049 283 416 T146G+H155 L (SEQ ID 2884 2424 34100 10026 248 4438 NO:218) 354 324 5312 1326 347 1060 N/A
L257G+H155 L
(SEQ ID 2364 1798 32044 9472 256 3690 NO:219) 691 368 5509 1812 363 1217 N/A
S283N+H155 L (SEQ ID 3162 2656 31504 9386 384 4014 NO:220) 1250 980 4414 1425 331 925 N/A
-KA:ent-kaurenoic acid -KL:ent-kaurenol Table 91.
Accumulation of steviol glycosides (in AUC) in a host comprising wild-type UGT76G1 or a UGT76G1 variant.
steviol+ steviol+ steviol+ steviol+ steviol+ steviol+ steviol+ steviol+
4G1c 5G1c 5G1c 5G1c 6G1c 6G1c 7G1c 7G1c (#33) (#22) (#24) (#25) (isomer (#23) (isomer (isomer 1) 2) 5) Wild-type (SEQ ID
7416 5230 1572 3622 7078 + 4474 NO:9) N/A N/A 1103 789 1044 590 912 (SEQ ID 122 + 7452 9450 320 1868 3894 +

NO:184) 345 N/A 2166 4068 905 825 1243 (SEQ ID 108 4382 3412 2792 4520 9388 + 4158 NO:181) N/A 305 1490 1176 1053 985 1677 (SEQ ID 114 3598 2996 3356 5438 10406 + 3700 NO:183) N/A 322 1630 745 1047 636 910 (SEQ ID 1158 754 842 1149 1429 1036 NO:185) (SEQ ID 4834 3358 3566 4350 9796 + 3924 NO:188) N/A N/A 1338 546 784 909 1619 Q23H+H

(SEQ ID 4468 3668 1932 3798 8764 + 3528 NO:217) N/A N/A 1172 679 380 619 1384 T146G+

(SEQ ID 3682 3008 2176 4022 8712 + 3284 NO:218) N/A N/A 1715 775 698 898 879 L257G+

(SEQ ID 3566 2974 956 2988 7046 + 3072 NO:219) N/A N/A 1693 781 1073 772 1660 S283N+

(SEQ ID 2670 2554 2430 3874 9450 + 2758 NO:220) N/A N/A 1807 444 1647 1837 3268 Example 9: Further characterization of UGT76G1 H155L Variant
[00229] UGT76G1 H155L (SEQ ID NO:184) was expressed in the steviol glycoside-producing S. cerevisiae strain described in Examples 2 and 8. As shown in Figure 6A, the strain expressing UGT76G1 H155L (gray bars) produced higher levels of RebM, RebA, RebB, 13-SMG, and rubusoside, compared to the control strain expressing wild-type UGT76G1 (black bars). The steviol glycoside-producing strain expressing UGT76G1 H155L
produced higher titers of RebM than RebD (Figure 6A).
[00230] The strain expressing UGT76G1 H155L (SEQ ID NO:184) produced greater total levels of steviol glycosides (13-SMG + 1,2-bioside + rubusoside + RebG + RebB
+ RebA +
RebE + RebD + RebM) and RebD + RebM (gray bars), compared to the control strain expressing wild-type UGT76G1 (black bars) (Figure 6B). Thus, the steviol glycoside-producing strain expressing UGT76G1 H155L (gray bars) demonstrated a 20% increase in steviol glycoside production and a 10% increase in RebD and RebM titers, compared to the control strain expressing wild-type UGT76G1 (black bars) (Figure 60).
[00231] The strain expressing UGT76G1 H155L (gray bars) also produced lesser amounts of a 1,2-bioside, 1,2-stevioside, a tri-glycosylated steviol molecule (stevio1+3GIc (#1)), a penta-glycosylated steviol molecule (stevio1+5GIc (#22), two hexa-glycosylated steviol molecules (stevio1+6GIc (isomer 1 and #23)), and a hepta-glycosylated steviol molecule (stevio1+7GIc (isomer 2)) but increased amounts of a tetra-glycosylated molecule (stevio1+4GIc (#26)) and two penta-glycosylated steviol molecules (Stevio1+5GIc (#24 and #25)), compared to the control strain expressing wild-type UGT76G1 (black bars) (Figure 6D). See Figures 1, 7, and 8 for structures of particular steviol glycosides detected.
[00232] Having described the invention in detail and by reference to specific embodiments thereof, it will be apparent that modifications and variations are possible without departing from the scope of the invention defined in the appended claims. More specifically, although some aspects of the present invention are identified herein as particularly advantageous, it is contemplated that the present invention is not necessarily limited to these particular aspects of the invention.
Table 10. Sequences disclosed herein.
SEQ ID NO:1 SEQ ID NO:2 SEQ ID NO:3 atggcagagc aacaaaagat caaaaagtca cctcacgtct tacttattcc atttcctctg 60 caaggacata tcaacccatt catacaattt gggaaaagat tgattagtaa gggtgtaaag 120 acaacactgg taaccactat ccacactttg aattctactc tgaaccactc aaatactact 180 actacaagta tagaaattca agctatatca gacggatgcg atgagggtgg ctttatgtct 240 gccggtgaat cttacttgga aacattcaag caagtgggat ccaagtctct ggccgatcta 300 atcaaaaagt tacagagtga aggcaccaca attgacgcca taatctacga ttctatgaca 360 gagtgggttt tagacgttgc tatcgaattt ggtattgatg gaggttcctt tttcacacaa 420 gcatgtgttg tgaattctct atactaccat gtgcataaag ggttaatctc tttaccattg 480 CA ()2973674 2017-07-12 ggtgaaactg tttcagttcc aggttttcca gtgttacaac gttgggaaac cccattgatc 540 ttacaaaatc atgaacaaat acaatcacct tggtcccaga tgttgtttgg tcaattcgct 600 aacatcgatc aagcaagatg ggtctttact aattcattct ataagttaga ggaagaggta 660 attgaatgga ctaggaagat ctggaatttg aaagtcattg gtccaacatt gccatcaatg 720 tatttggaca aaagacttga tgatgataaa gataatggtt tcaatttgta caaggctaat 780 catcacgaat gtatgaattg gctggatgac aaaccaaagg aatcagttgt atatgttgct 840 ttcggctctc ttgttaaaca tggtccagaa caagttgagg agattacaag agcacttata 900 gactctgacg taaacttttt gtgggtcatt aagcacaaag aggaggggaa actgccagaa 960 aacctttctg aagtgataaa gaccggaaaa ggtctaatcg ttgcttggtg taaacaattg 1020 gatgttttag ctcatgaatc tgtaggctgt tttgtaacac attgcggatt caactctaca 1080 ctagaagcca tttccttagg cgtacctgtc gttgcaatgc ctcagttctc cgatcagaca 1140 accaacgcta aacttttgga cgaaatacta ggggtgggtg tcagagttaa agcagacgag 1200 aatggtatcg tcagaagagg gaacctagct tcatgtatca aaatgatcat ggaagaggaa 1260 agaggagtta tcataaggaa aaacgcagtt aagtggaagg atcttgcaaa ggttgccgtc 1320 catgaaggcg gctcttcaga taatgatatt gttgaatttg tgtccgaact aatcaaagcc 1380 taa 1383 SEQ ID NO:4 SEQ ID NO:5 atggatgcaa tggctacaac tgagaagaaa ccacacgtca tcttcatacc atttccagca 60 caaagccaca ttaaagccat gctcaaacta gcacaacttc tccaccacaa aggactccag 120 ataaccttcg tcaacaccga cttcatccac aaccagtttc ttgaatcatc gggcccacat 180 tgtctagacg gtgcaccggg tttccggttc gaaaccattc cggatggtgt ttctcacagt 240 ccggaagcga gcatcccaat cagagaatca ctcttgagat ccattgaaac caacttcttg 300 gatcgtttca ttgatcttgt aaccaaactt ccggatcctc cgacttgtat tatctcagat 360 gggttcttgt cggttttcac aattgacgct gcaaaaaagc ttggaattcc ggtcatgatg 420 tattggacac ttgctgcctg tgggttcatg ggtttttacc atattcattc tctcattgag 480 aaaggatttg caccacttaa agatgcaagt tacttgacaa atgggtattt ggacaccgtc 540 attgattggg ttccgggaat ggaaggcatc cgtctcaagg atttcccgct ggactggagc 600 actgacctca atgacaaagt tttgatgttc actacggaag ctcctcaaag gtcacacaag 660 gtttcacatc atattttcca cacgttcgat gagttggagc ctagtattat aaaaactttg 720 tcattgaggt ataatcacat ttacaccatc ggcccactgc aattacttct tgatcaaata 780 cccgaagaga aaaagcaaac tggaattacg agtctccatg gatacagttt agtaaaagaa 840 gaaccagagt gtttccagtg gcttcagtct aaagaaccaa attccgtcgt ttatgtaaat 900 tttggaagta ctacagtaat gtctttagaa gacatgacgg aatttggttg gggacttgct 960 aatagcaacc attatttcct ttggatcatc cgatcaaact tggtgatagg ggaaaatgca 1020 gttttgcccc ctgaacttga ggaacatata aagaaaagag gctttattgc tagctggtgt 1080 tcacaagaaa aggtcttgaa gcacccttcg gttggagggt tcttgactca ttgtgggtgg 1140 ggatcgacca tcgagagctt gtctgctggg gtgccaatga tatgctggcc ttattcgtgg 1200 gaccagctga ccaactgtag gtatatatgc aaagaatggg aggttgggct cgagatggga 1260 accaaagtga aacgagatga agtcaagagg cttgtacaag agttgatggg agaaggaggt 1320 cacaaaatga ggaacaaggc taaagattgg aaagaaaagg ctcgcattgc aatagctcct 1380 aacggttcat cttctttgaa catagacaaa atggtcaagg aaatcaccgt gctagcaaga 1440 aactagttac aaagttgttt cacattgtgc tttctattta agatgtaact ttgttctaat 1500 ttaatattgt ctagatgtat tgaaccataa gtttagttgg tctcaggaat tgatttttaa 1560 tgaaataatg gtcattaggg gtgagt 1586 SEQ ID NO:6 CA ()2973674 2017-07-12 atggatgcaa tggcaactac tgagaaaaag cctcatgtga tcttcattcc atttcctgca 60 caatctcaca taaaggcaat gctaaagtta gcacaactat tacaccataa gggattacag 120 ataactttcg tgaataccga cttcatccat aatcaatttc tggaatctag tggccctcat 180 tgtttggacg gagccccagg gtttagattc gaaacaattc ctgacggtgt ttcacattcc 240 ccagaggcct ccatcccaat aagagagagt ttactgaggt caatagaaac caactttttg 300 gatcgtttca ttgacttggt cacaaaactt ccagacccac caacttgcat aatctctgat 360 ggctttctgt cagtgtttac tatcgacgct gccaaaaagt tgggtatccc agttatgatg 420 tactggactc ttgctgcatg cggtttcatg ggtttctatc acatccattc tcttatcgaa 480 aagggttttg ctccactgaa agatgcatca tacttaacca acggctacct ggatactgtt 540 attgactggg taccaggtat ggaaggtata agacttaaag attttccttt ggattggtct 600 acagacctta atgataaagt attgatgttt actacagaag ctccacaaag atctcataag 660 gtttcacatc atatctttca cacctttgat gaattggaac catcaatcat caaaaccttg 720 tctctaagat acaatcatat ctacactatt ggtccattac aattacttct agatcaaatt 780 cctgaagaga aaaagcaaac tggtattaca tccttacacg gctactcttt agtgaaagag 840 gaaccagaat gttttcaatg gctacaaagt aaagagccta attctgtggt ctacgtcaac 900 ttcggaagta caacagtcat gtccttggaa gatatgactg aatttggttg gggccttgct 960 aattcaaatc attactttct atggattatc aggtccaatt tggtaatagg ggaaaacgcc 1020 gtattacctc cagaattgga ggaacacatc aaaaagagag gtttcattgc ttcctggtgt 1080 tctcaggaaa aggtattgaa acatccttct gttggtggtt tccttactca ttgcggttgg 1140 ggctctacaa tcgaatcact aagtgcagga gttccaatga tttgttggcc atattcatgg 1200 gaccaactta caaattgtag gtatatctgt aaagagtggg aagttggatt agaaatggga 1260 acaaaggtta aacgtgatga agtgaaaaga ttggttcagg agttgatggg ggaaggtggc 1320 cacaagatga gaaacaaggc caaagattgg aaggaaaaag ccagaattgc tattgctcct 1380 aacgggtcat cctctctaaa cattgataag atggtcaaag agattacagt cttagccaga 1440 aactaa 1446 SEQ ID NO:7 SEQ ID NO:8 atggaaaaca agaccgaaac aacagttaga cgtaggcgta gaatcattct gtttccagta 60 ccttttcaag ggcacatcaa tccaatacta caactagcca acgttttgta ctctaaaggt 120 ttttctatta caatctttca caccaatttc aacaaaccaa aaacatccaa ttacccacat 180 ttcacattca gattcatact tgataatgat ccacaagatg aacgtatttc aaacttacct 240 acccacggtc ctttagctgg aatgagaatt ccaatcatca atgaacatgg tgccgatgag 300 cttagaagag aattagagtt acttatgttg gcatccgaag aggacgagga agtctcttgt 360 ctgattactg acgctctatg gtactttgcc caatctgtgg ctgatagttt gaatttgagg 420 agattggtac taatgacatc cagtctgttt aactttcacg ctcatgttag tttaccacaa 480 tttgacgaat tgggatactt ggaccctgat gacaagacta ggttagagga acaggcctct 540 ggttttccta tgttgaaagt caaagatatc aagtctgcct attctaattg gcaaatcttg 600 aaagagatct taggaaagat gatcaaacag acaaaggctt catctggagt gatttggaac 660 agtttcaaag agttagaaga gtctgaattg gagactgtaa tcagagaaat tccagcacct 720 tcattcctga taccattacc aaaacatttg actgcttcct cttcctcttt gttggatcat 780 gacagaacag tttttcaatg gttggaccaa caaccaccta gttctgtttt gtacgtgtca 840 tttggtagta cttctgaagt cgatgaaaag gacttccttg aaatcgcaag aggcttagtc 900 gatagtaagc agtcattcct ttgggtcgtg cgtccaggtt tcgtgaaagg ctcaacatgg 960 gtcgaaccac ttccagatgg ttttctaggc gaaagaggta gaatagtcaa atgggttcct 1020 caacaggaag ttttagctca tggcgctatt ggggcattct ggactcattc cggatggaat 1080 tcaactttag aatcagtatg cgaaggggta cctatgatct tttcagattt tggtcttgat 1140 CA ()2973674 2017-07-12 caaccactga acgcaagata catgtctgat gttttgaaag tgggtgtata tctagaaaat 1200 ggctgggaaa ggggtgaaat agctaatgca ataagacgtg ttatggttga tgaagagggg 1260 gagtatatca gacaaaacgc aagagtgctg aagcaaaagg ccgacgtttc tctaatgaag 1320 ggaggctctt catacgaatc cttagaatct cttgtttcct acatttcatc actgtaa 1377 SEQ ID NO:9 SEQ ID NO:10 atggctacat ctgattctat tgttgatgac aggaagcagt tgcatgtggc tactttccct 60 tggcttgctt tcggtcatat actgccttac ctacaactat caaaactgat agctgaaaaa 120 ggacataaag tgtcattcct ttcaacaact agaaacattc aaagattatc ttcccacata 180 tcaccattga ttaacgtcgt tcaattgaca cttccaagag tacaggaatt accagaagat 240 gctgaagcta caacagatgt gcatcctgaa gatatccctt acttgaaaaa ggcatccgat 300 ggattacagc ctgaggtcac tagattcctt gagcaacaca gtccagattg gatcatatac 360 gactacactc actattggtt gccttcaatt gcagcatcac taggcatttc tagggcacat 420 ttcagtgtaa ccacaccttg ggccattgct tacatgggtc catccgctga tgctatgatt 480 aacggcagtg atggtagaac taccgttgaa gatttgacaa ccccaccaaa gtggtttcca 540 tttccaacta aagtctgttg gagaaaacac gacttagcaa gactggttcc atacaaggca 600 ccaggaatct cagacggcta tagaatgggt ttagtcctta aagggtctga ctgcctattg 660 tctaagtgtt accatgagtt tgggacacaa tggctaccac ttttggaaac attacaccaa 720 gttcctgtcg taccagttgg tctattacct ccagaaatcc ctggtgatga gaaggacgag 780 acttgggttt caatcaaaaa gtggttagac gggaagcaaa aaggctcagt ggtatatgtg 840 gcactgggtt ccgaagtttt agtatctcaa acagaagttg tggaacttgc cttaggtttg 900 gaactatctg gattgccatt tgtctgggcc tacagaaaac caaaaggccc tgcaaagtcc 960 gattcagttg aattgccaga cggctttgtc gagagaacta gagatagagg gttggtatgg 1020 acttcatggg ctccacaatt gagaatcctg agtcacgaat ctgtgtgcgg tttcctaaca 1080 cattgtggtt ctggttctat agttgaagga ctgatgtttg gtcatccact tatcatgttg 1140 ccaatctttg gtgaccagcc tttgaatgca cgtctgttag aagataaaca agttggaatt 1200 gaaatcccac gtaatgagga agatggatgt ttaaccaagg agtctgtggc cagatcatta 1260 cgttccgttg tcgttgaaaa ggaaggcgaa atctacaagg ccaatgcccg tgaactttca 1320 aagatctaca atgacacaaa agtagagaag gaatatgttt ctcaatttgt agattaccta 1380 gagaaaaacg ctagagccgt agctattgat catgaatcct aa 1422 SEQ ID NO:11 SEQ ID NO:12 atggctactt ctgattccat cgttgacgat agaaagcaat tgcatgttgc tacttttcca 60 tggttggctt tcggtcatat tttgccatac ttgcaattgt ccaagttgat tgctgaaaag 120 ggtcacaagg tttcattctt gtctaccacc agaaacatcc aaagattgtc ctctcatatc 180 tccccattga tcaacgttgt tcaattgact ttgccaagag tccaagaatt gccagaagat 240 gctgaagcta ctactgatgt tcatccagaa gatatccctt acttgaaaaa ggcttccgat 300 SI:ON CH 03S

24404-25-25 44p-205-204g poqqabboab 04pp-2455-25 -254poabgoo 554pp-25505 OH' D4504-25-255 -205405p-25p -2005pp-2005 -2-20444545p PP05.205PPP bp-255-25545 OKI
baboqbpabq boqqababbo 5505045055 pababooabo 4450g-2554p babboabopp 00ZT -25p-2055455 pabqqabboo bopabpabab 5-2504p-2405 bababopabo pabbbpopab OtIT
abboggogab 005405g-20g pqqab000po 05504454pp gababbabog pop-25040pp 554055054o poop-25400g 4505055545 paboabopab abbqopgpab -25g-25-2040o OZOT
445554-25-25 D-250554504 5055050055 ababopabab -255-2504405 500500004o oqopaboabo -2500404505 54o-20005pp 55-24404055 5404004405 oboabhboab 006 gab-255405 bbogababog ababoppoqb 5p-25-25545p 5554o-2005g 55-2505-2055 Ot'8 -240505045o -24545045pp qbppoohboo bpababoabo gabbgaboog boo-2005Tel) bababboabb -2505005005 bp-254-2054g boaboabqpq goabbqqopq goo-244-24pp OZL bp-2455050o 4050-25045g opqaboopqb paelyabboab p.50445-2554 505405-255o 555045045o 4005-205-255 -25040405pp 544004044o bababoaboq 000454p-255 009 bog-204055p P.20050'24 -254mb-2-254p 55-25055455 -250445pp-2o ababbabbab Ot'g -200050-2555 pababoabqo bbabqoaboq babpaelyabb abababpboq abbaely20-25 08t' -205-2g-2004g bog-254-24p opabgogabb 5445445Tel) 4-2-205454-20 abghbppopo OZt' 5-25040405o aboabpaboo 5554o-20o-2o 0440450-25o 4504-204555 qoaboababq baboopabab 440445-255o 4044000505 005040555o p.504400555 -255=2004o 2504554pp phboabbpop boppoopoqb 0'25000'20 ambaboabab boaboopogo Ot'Z 5555-25045o 505000405o abgababbqb 044005045o gaboababog 050500005o 54550050pp 4005000g-2g popabbaboo 50-2004045o 4450454505 poppabbabo OZT
50450504pp bob-200050g opabogoabq 50004054pp -20055044pp 50405545pp 09 0540g-2545o 450-205g-255 5005005005 0050-24004o 04004o-2405 5004o-2554p 171:0N CH 03S
ELt' SEH
CIVAV?=1VN?lE qACAEOSAXE ?lEA?lIGNAfll SqE?:TVNV?lAI ESE=AAAal OZt' qaTVASE= 39CEENDIdIE ISA0?1(1=a1 VIT1,10(193Id gIATIqdHaaNg SEAIS9S9314 IqE93ASEHS MY-10(1VMSI 4AT-19?J(D:=E A39(IdgEASC S?1V(19?Id?DJA VMAZdq9SqE
00E '197d-lEAAEI OSArIVESSqV AAAAS9?10?19 CF1M=SAMI E=19dIEd dqq9AdAAdA
Ot'Z 01-FLIEggdgM OISZEHA3?1S 713(ISSWIAN SIADJASCSI9d V?lAdArnIVTI 1-DDI43A?Lid3 (13Mddid= EALDISCS9N INVCVSd9HA VIVMdIIASZ 14V?:ISIS'ISVV ISd'IMAHIAC
OZT
AIIMCdSHOE q,DILAEd0q9 CSV=AdIC EdHACIIVEV CEdgE0A?Idg IqOAANYIdS

IHSS'aTOII\DI IISqESA?IH9 ?lEVI'DISq0q AdqIHaaVqM dZIVAI-FIO?nl CCAISCSIVN
CI:ON CH 03S
ZZti ab 4404-2-254-20 Tabggpoabq 45405-25-24o 54p-25pp-2-25 bqqopqoabq 45044p-20pp 44450-24p-el) ppppaboqab P.200'24'250'2 pp-2404-25pp OZET
004544p-25p bpqaboppqo abppopqoqp -2-25455p-25p pp-2-254450g 544500g-25p 5444o4-25-24 05445404pp bpppoopfm. 054455g-25p -25p-254pp-el) P.20004'2'2'25 001 0g-2455045p pabppqabpp 554g-24g-25p qabopabqqp pop-2=2545 bqqqoqppoo OtIT 54454-244-25 4g-200o-2045 54445g-254g 455p-25445o T240445540 44554544pp 0801 go-2544044g 55454045pp gpabgpogog 54444p-25-el) qqppoppogo 55544044pp OZOT
5544455444 55-25-24-25-25 pqoppbppab 4450444554 abpoo544-2-2 54454044-25 096 4045p-2405p opq55-2-2-240 OPPPP5P0Pq 4055544454 4g-200544mb 5404544p-25 5444555444 05544p-254g 5445p-250pp -2-204044455 qqqabpaboo 4455544405 Ot'8 445g-24445g 454044555p pp-205-2-2455 T255445545 PPPPPOT200 44455544pp PaboabPPPP -250-25455pp oqqppabpoo poo5445444 bb boo5445-200445 20g-20544p 0pp-255445g qqoabqq&bq ppoqopqhbo 44p-25g-204p 40545p-240g 5445qqabgq abooqqabpp -254444554p 45554-2-25-20 -24455g-254o 444-2455poo qabpppopqp poqq&bqqab pqabbqqqab 0'205Pb 54454045pp -2pp-2-20044g Ot'g -20044455mb P.2'200'20040 pqoabqqqab -2-254450o-2g op-25-24554p 54044550pp 08t' qqabqpqabq -25405404pp 045554-24-24 qabqqpqabb 5g-2004o-24o -2445404044 OZt' T20005-25-24 044g-24555g T204405405 4g-200g-2005 44554o-244p oqopqpqqab opqogpogab 54g-25-2000o qqpoppopab bqqoqqabpq 0-2445p-25pp op-20-244455 LOOZSO/9IOLI1LL3c1 98t0Z1/9I0ZCOA

CA ()2973674 2017-07-12 atggatagtg gctactcctc atcttatgct gctgccgctg gtatgcacgt tgtgatctgc 60 ccttggttgg cctttggtca cctgttacca tgtctggatt tagcccaaag actggcctca 120 agaggccata gagtatcatt tgtgtctact cctagaaata tctctcgttt accaccagtc 180 agacctgctc tagctcctct agttgcattc gttgctcttc cacttccaag agtagaagga 240 ttgccagacg gcgctgaatc tactaatgac gtaccacatg atagacctga catggtcgaa 300 ttgcatagaa gagcctttga tggattggca gctccatttt ctgagttcct gggcacagca 360 tgtgcagact gggttatagt cgatgtattt catcactggg ctgctgcagc cgcattggaa 420 cataaggtgc cttgtgctat gatgttgtta gggtcagcac acatgatcgc atccatagct 480 gatagaagat tggaaagagc tgaaacagaa tccccagccg cagcaggaca aggtaggcca 540 gctgccgccc caacctttga agtggctaga atgaaattga ttcgtactaa aggtagttca 600 gggatgagtc ttgctgaaag gttttctctg acattatcta gatcatcatt agttgtaggt 660 agatcctgcg tcgagttcga acctgaaaca gtacctttac tatctacttt gagaggcaaa 720 cctattactt tccttggtct aatgcctcca ttacatgaag gaaggagaga agatggtgaa 780 gatgctactg ttaggtggtt agatgcccaa cctgctaagt ctgttgttta cgttgcattg 840 ggttctgagg taccactagg ggtggaaaag gtgcatgaat tagcattagg acttgagctg 900 gccggaacaa gattcctttg ggctttgaga aaaccaaccg gtgtttctga cgccgacttg 960 ctaccagctg ggttcgaaga gagaacaaga ggccgtggtg tcgttgctac tagatgggtc 1020 ccacaaatga gtattctagc tcatgcagct gtaggggcct ttctaaccca ttgcggttgg 1080 aactcaacaa tagaaggact gatgtttggt catccactta ttatgttacc aatctttggc 1140 gatcagggac ctaacgcaag attgattgag gcaaagaacg caggtctgca ggttgcacgt 1200 aatgatggtg atggttcctt tgatagagaa ggcgttgcag ctgccatcag agcagtcgcc 1260 gttgaggaag agtcatctaa agttttccaa gctaaggcca aaaaattaca agagattgtg 1320 gctgacatgg cttgtcacga aagatacatc gatggtttca tccaacaatt gagaagttat 1380 aaagactaa 1389 SEQ ID NO:16 SEQ ID NO:17 SEQ ID NO:18 SEQ ID NO:19 atggctttgg taaacccaac cgctcttttc tatggtacct ctatcagaac aagacctaca 60 CA ()2973674 2017-07-12 aacttactaa atccaactca aaagctaaga ccagtttcat catcttcctt accttctttc 120 tcatcagtta gtgcgattct tactgaaaaa catcaatcta atccttctga gaacaacaat 180 ttgcaaactc atctagaaac tcctttcaac tttgatagtt atatgttgga aaaagtcaac 240 atggttaacg aggcgcttga tgcatctgtc ccactaaaag acccaatcaa aatccatgaa 300 tccatgagat actctttatt ggcaggcggt aagagaatca gaccaatgat gtgtattgca 360 gcctgcgaaa tagtcggagg taatatcctt aacgccatgc cagccgcatg tgccgtggaa 420 atgattcata ctatgtcttt ggtgcatgac gatcttccat gtatggataa tgatgacttc 480 agaagaggta aacctatttc acacaaggtc tacggggagg aaatggcagt attgaccggc 540 gatgctttac taagtttatc tttcgaacat atagctactg ctacaaaggg tgtatcaaag 600 gatagaatcg tcagagctat aggggagttg gcccgttcag ttggctccga aggtttagtg 660 gctggacaag ttgtagatat cttgtcagag ggtgctgatg ttggattaga tcacctagaa 720 tacattcaca tccacaaaac agcaatgttg cttgagtcct cagtagttat tggcgctatc 780 atgggaggag gatctgatca gcagatcgaa aagttgagaa aattcgctag atctattggt 840 ctactattcc aagttgtgga tgacattttg gatgttacaa aatctaccga agagttgggg 900 aaaacagctg gtaaggattt gttgacagat aagacaactt acccaaagtt gttaggtata 960 gaaaagtcca gagaatttgc cgaaaaactt aacaaggaag cacaagagca attaagtggc 1020 tttgatagac gtaaggcagc tcctttgatc gcgttagcca actacaatgc gtaccgtcaa 1080 aattga 1086 SEQ ID NO:20 SEQ ID NO:21 atggctgagc aacaaatatc taacttgctg tctatgtttg atgcttcaca tgctagtcag 60 aaattagaaa ttactgtcca aatgatggac acataccatt acagagaaac gcctccagat 120 tcctcatctt ctgaaggcgg ttcattgtct agatacgacg agagaagagt ctctttgcct 180 ctcagtcata atgctgcctc tccagatatt gtatcacaac tatgtttttc cactgcaatg 240 tcttcagagt tgaatcacag atggaaatct caaagattaa aggtggccga ttctccttac 300 aactatatcc taacattacc atcaaaagga attagaggtg cctttatcga ttccctgaac 360 gtatggttgg aggttccaga ggatgaaaca tcagtcatca aggaagttat tggtatgctc 420 cacaactctt cattaatcat tgatgacttc caagataatt ctccacttag aagaggaaag 480 ccatctaccc atacagtctt cggccctgcc caggctatca atactgctac ttacgttata 540 gttaaagcaa tcgaaaagat acaagacata gtgggacacg atgcattggc agatgttacg 600 ggtactatta caactatttt ccaaggtcag gccatggact tgtggtggac agcaaatgca 660 atcgttccat caatacagga atacttactt atggtaaacg ataaaaccgg tgctctcttt 720 agactgagtt tggagttgtt agctctgaat tccgaagcca gtatttctga ctctgcttta 780 gaaagtttat ctagtgctgt ttccttgcta ggtcaatact tccaaatcag agacgactat 840 atgaacttga tcgataacaa gtatacagat cagaaaggct tctgcgaaga tcttgatgaa 900 ggcaagtact cactaacact tattcatgcc ctccaaactg attcatccga tctactgacc 960 aacatccttt caatgagaag agtgcaagga aagttaacgg cacaaaagag atgttggttc 1020 tggaaatga 1029 SEQ ID NO:22 SEQ ID NO:23 CA ()2973674 2017-07-12 atggaaaaga ctaaggagaa agcagaacgt atcttgctgg agccatacag atacttatta 60 caactaccag gaaagcaagt ccgttctaaa ctatcacaag cgttcaatca ctggttaaaa 120 gttcctgaag ataagttaca aatcattatt gaagtcacag aaatgctaca caatgcttct 180 ttactgatcg atgatataga ggattcttcc aaactgagaa gaggttttcc tgtcgctcat 240 tccatatacg gggtaccaag tgtaatcaac tcagctaatt acgtctactt cttgggattg 300 gaaaaagtat tgacattaga tcatccagac gctgtaaagc tattcaccag acaacttctt 360 gaattgcatc aaggtcaagg tttggatatc tattggagag acacttatac ttgcccaaca 420 gaagaggagt acaaagcaat ggttctacaa aagactggcg gtttgttcgg acttgccgtt 480 ggtctgatgc aacttttctc tgattacaag gaggacttaa agcctctgtt ggataccttg 540 ggcttgtttt tccagattag agatgactac gctaacttac attcaaagga atattcagaa 600 aacaaatcat tctgtgaaga tttgactgaa gggaagttta gttttccaac aatccacgcc 660 atttggtcaa gaccagaatc tactcaagtg caaaacattc tgcgtcagag aacagagaat 720 attgacatca aaaagtattg tgttcagtac ttggaagatg ttggttcttt tgcttacaca 780 agacatacac ttagagaatt agaggcaaaa gcatacaagc aaatagaagc ctgtggaggc 840 aatccttctc tagtggcatt ggttaaacat ttgtccaaaa tgttcaccga ggaaaacaag 900 taa 903 SEQ ID NO:24 SEQ ID NO:25 atggcaagat tctattttct taacgcacta ttgatggtta tctcattaca atcaactaca 60 gccttcactc cagctaaact tgcttatcca acaacaacaa cagctctaaa tgtcgcctcc 120 gccgaaactt ctttcagtct agatgaatac ttggcctcta agataggacc tatagagtct 180 gccttggaag catcagtcaa atccagaatt ccacagaccg ataagatctg cgaatctatg 240 gcctactctt tgatggcagg aggcaagaga attagaccag tgttgtgtat cgctgcatgt 300 gagatgttcg gtggatccca agatgtcgct atgcctactg ctgtggcatt agaaatgata 360 cacacaatgt ctttgattca tgatgatttg ccatccatgg ataacgatga cttgagaaga 420 ggtaaaccaa caaaccatgt cgttttcggc gaagatgtag ctattcttgc aggtgactct 480 ttattgtcaa cttccttcga gcacgtcgct agagaaacaa aaggagtgtc agcagaaaag 540 atcgtggatg ttatcgctag attaggcaaa tctgttggtg ccgagggcct tgctggcggt 600 caagttatgg acttagaatg tgaagctaaa ccaggtacca cattagacga cttgaaatgg 660 attcatatcc ataaaaccgc tacattgtta caagttgctg tagcttctgg tgcagttcta 720 ggtggtgcaa ctcctgaaga ggttgctgca tgcgagttgt ttgctatgaa tataggtctt 780 gcctttcaag ttgccgacga tatccttgat gtaaccgctt catcagaaga tttgggtaaa 840 actgcaggca aagatgaagc tactgataag acaacttacc caaagttatt aggattagaa 900 gagagtaagg catacgcaag acaactaatc gatgaagcca aggaaagttt ggctcctttt 960 ggagatagag ctgccccttt attggccatt gcagatttca ttattgatag aaagaattga 1020 SEQ ID NO:26 SEQ ID NO:27 atgcacttag caccacgtag agtccctaga ggtagaagat caccacctga cagagttcct 60 gaaagacaag gtgccttggg tagaagacgt ggagctggct ctactggctg tgcccgtgct 120 gctgctggtg ttcaccgtag aagaggagga ggcgaggctg atccatcagc tgctgtgcat 180 agaggctggc aagccggtgg tggcaccggt ttgcctgatg aggtggtgtc taccgcagcc 240 gccttagaaa tgtttcatgc ttttgcttta atccatgatg atatcatgga tgatagtgca 300 CA ()2973674 2017-07-12 actagaagag gctccccaac tgttcacaga gccctagctg atcgtttagg cgctgctctg 360 gacccagatc aggccggtca actaggagtt tctactgcta tcttggttgg agatctggct 420 ttgacatggt ccgatgaatt gttatacgct ccattgactc cacatagact ggcagcagta 480 ctaccattgg taacagctat gagagctgaa accgttcatg gccaatatct tgatataact 540 agtgctagaa gacctgggac cgatacttct cttgcattga gaatagccag atataagaca 600 gcagcttaca caatggaacg tccactgcac attggtgcag ccctggctgg ggcaagacca 660 gaactattag cagggctttc agcatacgcc ttgccagctg gagaagcctt ccaattggca 720 gatgacctgc taggcgtctt cggtgatcca agacgtacag ggaaacctga cctagatgat 780 cttagaggtg gaaagcatac tgtcttagtc gccttggcaa gagaacatgc cactccagaa 840 cagagacaca cattggatac attattgggt acaccaggtc ttgatagaca aggcgcttca 900 agactaagat gcgtattggt agcaactggt gcaagagccg aagccgaaag acttattaca 960 gagagaagag atcaagcatt aactgcattg aacgcattaa cactgccacc tcctttagct 1020 gaggcattag caagattgac attagggtct acagctcatc ctgcctaa 1068 SEQ ID NO:28 SEQ ID NO:29 atgtcatatt tcgataacta cttcaatgag atagttaatt ccgtgaacga catcattaag 60 tcttacatct ctggcgacgt accaaaacta tacgaagcct cctaccattt gtttacatca 120 ggaggaaaga gactaagacc attgatcctt acaatttctt ctgatctttt cggtggacag 180 agagaaagag catactatgc tggcgcagca atcgaagttt tgcacacatt cactttggtt 240 cacgatgata tcatggatca agataacatt cgtagaggtc ttcctactgt acatgtcaag 300 tatggcctac ctttggccat tttagctggt gacttattgc atgcaaaagc ctttcaattg 360 ttgactcagg cattgagagg tctaccatct gaaactatca tcaaggcgtt tgatatcttt 420 acaagatcta tcattatcat atcagaaggt caagctgtcg atatggaatt cgaagataga 480 attgatatca aggaacaaga gtatttggat atgatatctc gtaaaaccgc tgccttattc 540 tcagcttctt cttccattgg ggcgttgata gctggagcta atgataacga tgtgagatta 600 atgtccgatt tcggtacaaa tcttgggatc gcatttcaaa ttgtagatga tatacttggt 660 ttaacagctg atgaaaaaga gctaggaaaa cctgttttca gtgatatcag agaaggtaaa 720 aagaccatat tagtcattaa gactttagaa ttgtgtaagg aagacgagaa aaagattgtg 780 ttaaaagcgc taggcaacaa gtcagcatca aaggaagagt tgatgagttc tgctgacata 840 atcaaaaagt actcattgga ttacgcctac aacttagctg agaaatacta caaaaacgcc 900 atcgattctc taaatcaagt ttcaagtaaa agtgatattc cagggaaggc attgaaatat 960 cttgctgaat tcaccatcag aagacgtaag taa 993 SEQ ID NO:30 SEQ ID NO:31 atggtcgcac aaactttcaa cctggatacc tacttatccc aaagacaaca acaagttgaa 60 gaggccctaa gtgctgctct tgtgccagct tatcctgaga gaatatacga agctatgaga 120 tactccctcc tggcaggtgg caaaagatta agacctatct tatgtttagc tgcttgcgaa 180 ttggcaggtg gttctgttga acaagccatg ccaactgcgt gtgcacttga aatgatccat 240 acaatgtcac taattcatga tgacctgcca gccatggata acgatgattt cagaagagga 300 aagccaacta atcacaaggt gttcggggaa gatatagcca tcttagcggg tgatgcgctt 360 ttagcttacg cttttgaaca tattgcttct caaacaagag gagtaccacc tcaattggtg 420 CA ()2973674 2017-07-12 ctacaagtta ttgctagaat cggacacgcc gttgctgcaa caggcctcgt tggaggccaa 480 gtcgtagacc ttgaatctga aggtaaagct atttccttag aaacattgga gtatattcac 540 tcacataaga ctggagcctt gctggaagca tcagttgtct caggcggtat tctcgcaggg 600 gcagatgaag agcttttggc cagattgtct cattacgcta gagatatagg cttggctttt 660 caaatcgtcg atgatatcct ggatgttact gctacatctg aacagttggg gaaaaccgct 720 ggtaaagacc aggcagccgc aaaggcaact tatccaagtc tattgggttt agaagcctct 780 agacagaaag cggaagagtt gattcaatct gctaaggaag ccttaagacc ttacggttca 840 caagcagagc cactcctagc gctggcagac ttcatcacac gtcgtcagca ttaa 894 SEQ ID NO:32 SEQ ID NO:33 atgaaaaccg ggtttatctc accagcaaca gtatttcatc acagaatctc accagcgacc 60 actttcagac atcacttatc acctgctact acaaactcta caggcattgt cgccttaaga 120 gacatcaact tcagatgtaa agcagtttct aaagagtact ctgatctgtt gcagaaagat 180 gaggcttctt tcacaaaatg ggacgatgac aaggtgaaag atcatcttga taccaacaaa 240 aacttatacc caaatgatga gattaaggaa tttgttgaat cagtaaaggc tatgttcggt 300 agtatgaatg acggggagat aaacgtctct gcatacgata ctgcatgggt tgctttggtt 360 caagatgtcg atggatcagg tagtcctcag ttcccttctt ctttagaatg gattgccaac 420 aatcaattgt cagatggatc atggggagat catttgctgt tctcagctca cgatagaatc 480 atcaacacat tagcatgcgt tattgcactt acaagttgga atgttcatcc ttctaagtgt 540 gaaaaaggtt tgaattttct gagagaaaac atttgcaaat tagaagatga aaacgcagaa 600 catatgccaa ttggttttga agtaacattc ccatcactaa ttgatatcgc gaaaaagttg 660 aacattgaag tacctgagga tactccagca cttaaagaga tctacgcacg tagagatatc 720 aagttaacta agatcccaat ggaagttctt cacaaggtac ctactacttt gttacattct 780 ttggaaggaa tgcctgattt ggagtgggaa aaactgttaa agctacaatg taaagatggt 840 agtttcttgt tttccccatc tagtaccgca ttcgccctaa tgcaaacaaa agatgagaaa 900 tgcttacagt atctaacaaa tatcgtcact aagttcaacg gtggcgtgcc taatgtgtac 960 ccagtcgatt tgtttgaaca tatttgggtt gttgatagac tgcagagatt ggggattgcc 1020 agatacttca aatcagagat aaaagattgt gtagagtata tcaataagta ctggaccaaa 1080 aatggaattt gttgggctag aaatactcac gttcaagata tcgatgatac agccatggga 1140 ttcagagtgt tgagagcgca cggttatgac gtcactccag atgtttttag acaatttgaa 1200 aaagatggta aattcgtttg ctttgcaggg caatcaacac aagccgtgac aggaatgttt 1260 aacgtttaca gagcctctca aatgttgttc ccaggggaga gaattttgga agatgccaaa 1320 aagttctctt acaattactt aaaggaaaag caaagtacca acgaattgct ggataaatgg 1380 ataatcgcta aagatctacc tggtgaagtt ggttatgctc tggatatccc atggtatgct 1440 tccttaccaa gattggaaac tcgttattac cttgaacaat acggcggtga agatgatgtc 1500 tggataggca agacattata cagaatgggt tacgtgtcca ataacacata tctagaaatg 1560 gcaaagctgg attacaataa ctatgttgca gtccttcaat tagaatggta cacaatacaa 1620 caatggtacg tcgatattgg tatagagaag ttcgaatctg acaacatcaa gtcagtcctg 1680 SEQ ID NO:34 CA ()2973674 2017-07-12 SEQ ID NO:35 atgcctgatg cacacgatgc tccacctcca caaataagac agagaacact agtagatgag 60 gctacccaac tgctaactga gtccgcagaa gatgcatggg gtgaagtcag tgtgtcagaa 120 tacgaaacag caaggctagt tgcccatgct acatggttag gtggacacgc cacaagagtg 180 gccttccttc tggagagaca acacgaagac gggtcatggg gtccaccagg tggatatagg 240 ttagtcccta cattatctgc tgttcacgca ttattgacat gtcttgcctc tcctgctcag 300 gatcatggcg ttccacatga tagactttta agagctgttg acgcaggctt gactgccttg 360 agaagattgg ggacatctga ctccccacct gatactatag cagttgagct ggttatccca 420 tctttgctag agggcattca acacttactg gaccctgctc atcctcatag tagaccagcc 480 ttctctcaac atagaggctc tcttgtttgt cctggtggac tagatgggag aactctagga 540 gctttgagat cacacgccgc agcaggtaca ccagtaccag gaaaagtctg gcacgcttcc 600 gagactttgg gcttgagtac cgaagctgct tctcacttgc aaccagccca aggtataatc 660 ggtggctctg ctgctgccac agcaacatgg ctaaccaggg ttgcaccatc tcaacagtca 720 gattctgcca gaagatacct tgaggaatta caacacagat actctggccc agttccttcc 780 attaccccta tcacatactt cgaaagagca tggttattga acaattttgc agcagccggt 840 gttccttgtg aggctccagc tgctttgttg gattccttag aagcagcact tacaccacaa 900 ggtgctcctg ctggagcagg attgcctcca gatgctgatg atacagccgc tgtgttgctt 960 gcattggcaa cacatgggag aggtagaaga ccagaagtac tgatggatta caggactgac 1020 gggtatttcc aatgctttat tggggaaagg actccatcaa tttcaacaaa cgctcacgta 1080 ttggaaacat tagggcatca tgtggcccaa catccacaag atagagccag atacggatca 1140 gccatggata ccgcatcagc ttggctgctg gcagctcaaa agcaagatgg ctcttggtta 1200 gataaatggc atgcctcacc atactacgct actgtttgtt gcacacaagc cctagccgct 1260 catgcaagtc ctgcaactgc accagctaga cagagagctg tcagatgggt tttagccaca 1320 caaagatccg atggcggttg gggtctatgg cattcaactg ttgaagagac tgcttatgcc 1380 ttacagatct tggccccacc ttctggtggt ggcaatatcc cagtccaaca agcacttact 1440 agaggcagag caagattgtg tggagccttg ccactgactc ctttatggca tgataaggat 1500 ttgtatactc cagtaagagt agtcagagct gccagagctg ctgctctgta cactaccaga 1560 gatctattgt taccaccatt gtaa 1584 SEQ ID NO:36 SEQ ID NO:37 atgaacgccc tatccgaaca cattttgtct gaattgagaa gattattgtc tgaaatgagt 60 gatggcggat ctgttggtcc atctgtgtat gatacggccc aggccctaag attccacggt 120 aacgtaacag gtagacaaga tgcatatgct tggttgatcg cccagcaaca agcagatgga 180 ggttggggct ctgccgactt tccactcttt agacatgctc caacatgggc tgcacttctc 240 gcattacaaa gagctgatcc acttcctggc gcagcagacg cagttcagac cgcaacaaga 300 ttcttgcaaa gacaaccaga tccatacgct catgccgttc ctgaggatgc ccctattggt 360 gctgaactga tcttgcctca gttttgtgga gaggctgctt ggttgttggg aggtgtggcc 420 ttccctagac acccagccct attaccatta agacaggctt gtttagtcaa actgggtgca 480 gtcgccatgt tgccttcagg acacccattg ctccactcct gggaggcatg gggtacttct 540 ccaacaacag cctgtccaga cgatgatggt tctataggta tctcaccagc agctacagcc 600 gcctggagag cccaggctgt gaccagaggc tcaactcctc aagtgggcag agctgacgca 660 CA ()2973674 2017-07-12 tacttacaaa tggcttcaag agcaacgaga tcaggcatag aaggagtctt ccctaatgtt 720 tggcctataa acgtattcga accatgctgg tcactgtaca ctctccatct tgccggtctg 780 ttcgcccatc cagcactggc tgaggctgta agagttatcg ttgctcaact tgaagcaaga 840 ttgggagtgc atggcctcgg accagcttta cattttgctg ccgacgctga tgatactgca 900 gttgccttat gcgttctgca tttggctggc agagatcctg cagttgacgc attgagacat 960 tttgaaattg gtgagctctt tgttacattc ccaggagaga gaaatgctag tgtctctacg 1020 aacattcacg ctcttcatgc tttgagattg ttaggtaaac cagctgccgg agcaagtgca 1080 tacgtcgaag caaatagaaa tccacatggt ttgtgggaca acgaaaaatg gcacgtttca 1140 tggctttatc caactgcaca cgccgttgca gctctagctc aaggcaagcc tcaatggaga 1200 gatgaaagag cactagccgc tctactacaa gctcaaagag atgatggtgg ttggggagct 1260 ggtagaggat ccactttcga ggaaaccgcc tacgctcttt tcgctttaca cgttatggac 1320 ggatctgagg aagccacagg cagaagaaga atcgctcaag tcgtcgcaag agccttagaa 1380 tggatgctag ctagacatgc cgcacatgga ttaccacaaa caccactctg gattggtaag 1440 gaattgtact gtcctactag agtcgtaaga gtagctgagc tagctggcct gtggttagca 1500 ttaagatggg gtagaagagt attagctgaa ggtgctggtg ctgcacctta a 1551 SEQ ID NO:38 SEQ ID NO:39 atggttttgt cttcttcttg tactacagta ccacacttat cttcattagc tgtcgtgcaa 60 cttggtcctt ggagcagtag gattaaaaag aaaaccgata ctgttgcagt accagccgct 120 gcaggaaggt ggagaagggc cttggctaga gcacagcaca catcagaatc cgcagctgtc 180 gcaaagggca gcagtttgac ccctatagtg agaactgacg ctgagtcaag gagaacaaga 240 tggccaaccg atgacgatga cgccgaacct ttagtggatg agatcagggc aatgcttact 300 tccatgtctg atggtgacat ttccgtgagc gcatacgata cagcctgggt cggattggtt 360 ccaagattag acggcggtga aggtcctcaa tttccagcag ctgtgagatg gataagaaat 420 aaccagttgc ctgacggaag ttggggcgat gccgcattat tctctgccta tgacaggctt 480 atcaataccc ttgcctgcgt tgtaactttg acaaggtggt ccctagaacc agagatgaga 540 ggtagaggac tatctttttt gggtaggaac atgtggaaat tagcaactga agatgaagag 600 tcaatgccta ttggcttcga attagcattt ccatctttga tagagcttgc taagagccta 660 ggtgtccatg acttccctta tgatcaccag gccctacaag gaatctactc ttcaagagag 720 atcaaaatga agaggattcc aaaagaagtg atgcataccg ttccaacatc aatattgcac 780 agtttggagg gtatgcctgg cctagattgg gctaaactac ttaaactaca gagcagcgac 840 ggaagttttt tgttctcacc agctgccact gcatatgctt taatgaatac cggagatgac 900 aggtgtttta gctacatcga tagaacagta aagaaattca acggcggcgt ccctaatgtt 960 tatccagtgg atctatttga acatatttgg gccgttgata gacttgaaag attaggaatc 1020 tccaggtact tccaaaagga gatcgaacaa tgcatggatt atgtaaacag gcattggact 1080 gaggacggta tttgttgggc aaggaactct gatgtcaaag aggtggacga cacagctatg 1140 gcctttagac ttcttaggtt gcacggctac agcgtcagtc ctgatgtgtt taaaaacttc 1200 gaaaaggacg gtgaattttt cgcatttgtc ggacagtcta atcaagctgt taccggtatg 1260 tacaacttaa acagagcaag ccagatatcc ttcccaggcg aggatgtgct tcatagagct 1320 ggtgccttct catatgagtt cttgaggaga aaagaagcag agggagcttt gagggacaag 1380 tggatcattt ctaaagatct acctggtgaa gttgtgtata ctttggattt tccatggtac 1440 ggcaacttac ctagagtcga ggccagagac tacctagagc aatacggagg tggtgatgac 1500 gtttggattg gcaagacatt gtataggatg ccacttgtaa acaatgatgt atatttggaa 1560 ttggcaagaa tggatttcaa ccactgccag gctttgcatc agttagagtg gcaaggacta 1620 aaaagatggt atactgaaaa taggttgatg gactttggtg tcgcccaaga agatgccctt 1680 agagcttatt ttcttgcagc cgcatctgtt tacgagcctt gtagagctgc cgagaggctt 1740 CA ()2973674 2017-07-12 gcatgggcta gagccgcaat actagctaac gccgtgagca cccacttaag aaatagccca 1800 tcattcagag aaaggttaga gcattctctt aggtgtagac ctagtgaaga gacagatggc 1860 tcctggttta actcctcaag tggctctgat gcagttttag taaaggctgt cttaagactt 1920 actgattcat tagccaggga agcacagcca atccatggag gtgacccaga agatattata 1980 cacaagttgt taagatctgc ttgggccgag tgggttaggg aaaaggcaga cgctgccgat 2040 agcgtgtgca atggtagttc tgcagtagaa caagagggat caagaatggt ccatgataaa 2100 cagacctgtc tattattggc tagaatgatc gaaatttctg ccggtagggc agctggtgaa 2160 gcagccagtg aggacggcga tagaagaata attcaattaa caggctccat ctgcgacagt 2220 cttaagcaaa aaatgctagt ttcacaggac cctgaaaaaa atgaagagat gatgtctcac 2280 gtggatgacg aattgaagtt gaggattaga gagttcgttc aatatttgct tagactaggt 2340 gaaaaaaaga ctggatctag cgaaaccagg caaacatttt taagtatagt gaaatcatgt 2400 tactatgctg ctcattgccc acctcatgtc gttgatagac acattagtag agtgattttc 2460 gagccagtaa gtgccgcaaa gtaaccgcgg 2490 SEQ ID NO:40 SEQ ID NO:41 cttcttcact aaatacttag acagagaaaa cagagctttt taaagccatg tctcttcagt 60 atcatgttct aaactccatt ccaagtacaa cctttctcag ttctactaaa acaacaatat 120 cttcttcttt ccttaccatc tcaggatctc ctctcaatgt cgctagagac aaatccagaa 180 gcggttccat acattgttca aagcttcgaa ctcaagaata cattaattct caagaggttc 240 aacatgattt gcctctaata catgagtggc aacagcttca aggagaagat gctcctcaga 300 ttagtgttgg aagtaatagt aatgcattca aagaagcagt gaagagtgtg aaaacgatct 360 tgagaaacct aacggacggg gaaattacga tatcggctta cgatacagct tgggttgcat 420 tgatcgatgc cggagataaa actccggcgt ttccctccgc cgtgaaatgg atcgccgaga 480 accaactttc cgatggttct tggggagatg cgtatctctt ctcttatcat gatcgtctca 540 tcaataccct tgcatgcgtc gttgctctaa gatcatggaa tctctttcct catcaatgca 600 acaaaggaat cacgtttttc cgggaaaata ttgggaagct agaagacgaa aatgatgagc 660 atatgccaat cggattcgaa gtagcattcc catcgttgct tgagatagct cgaggaataa 720 acattgatgt accgtacgat tctccggtct taaaagatat atacgccaag aaagagctaa 780 agcttacaag gataccaaaa gagataatgc acaagatacc aacaacattg ttgcatagtt 840 tggaggggat gcgtgattta gattgggaaa agctcttgaa acttcaatct caagacggat 900 ctttcctctt ctctccttcc tctaccgctt ttgcattcat gcagacccga gacagtaact 960 gcctcgagta tttgcgaaat gccgtcaaac gtttcaatgg aggagttccc aatgtctttc 1020 ccgtggatct tttcgagcac atatggatag tggatcggtt acaacgttta gggatatcga 1080 gatactttga agaagagatt aaagagtgtc ttgactatgt ccacagatat tggaccgaca 1140 atggcatatg ttgggctaga tgttcccatg tccaagacat cgatgataca gccatggcat 1200 ttaggctctt aagacaacat ggataccaag tgtccgcaga tgtattcaag aactttgaga 1260 aagagggaga gtttttctgc tttgtggggc aatcaaacca agcagtaacc ggtatgttca 1320 acctataccg ggcatcacaa ttggcgtttc caagggaaga gatattgaaa aacgccaaag 1380 agttttctta taattatctg ctagaaaaac gggagagaga ggagttgatt gataagtgga 1440 ttataatgaa agacttacct ggcgagattg ggtttgcgtt agagattcca tggtacgcaa 1500 gcttgcctcg agtagagacg agattctata ttgatcaata tggtggagaa aacgacgttt 1560 CA ()2973674 2017-07-12 ggattggcaa gactctttat aggatgccat acgtgaacaa taatggatat ctggaattag 1620 caaaacaaga ttacaacaat tgccaagctc agcatcagct cgaatgggac atattccaaa 1680 agtggtatga agaaaatagg ttaagtgagt ggggtgtgcg cagaagtgag cttctcgagt 1740 gttactactt agcggctgca actatatttg aatcagaaag gtcacatgag agaatggttt 1800 gggctaagtc aagtgtattg gttaaagcca tttcttcttc ttttggggaa tcctctgact 1860 ccagaagaag cttctccgat cagtttcatg aatacattgc caatgctcga cgaagtgatc 1920 atcactttaa tgacaggaac atgagattgg accgaccagg atcggttcag gccagtcggc 1980 ttgccggagt gttaatcggg actttgaatc aaatgtcttt tgaccttttc atgtctcatg 2040 gccgtgacgt taacaatctc ctctatctat cgtggggaga ttggatggaa aaatggaaac 2100 tatatggaga tgaaggagaa ggagagctca tggtgaagat gataattcta atgaagaaca 2160 atgacctaac taacttcttc acccacactc acttcgttcg tctcgcggaa atcatcaatc 2220 gaatctgtct tcctcgccaa tacttaaagg caaggagaaa cgatgagaag gagaagacaa 2280 taaagagtat ggagaaggag atggggaaaa tggttgagtt agcattgtcg gagagtgaca 2340 catttcgtga cgtcagcatc acgtttcttg atgtagcaaa agcattttac tactttgctt 2400 tatgtggcga tcatctccaa actcacatct ccaaagtctt gtttcaaaaa gtctagtaac 2460 ctcatcatca tcatcgatcc attaacaatc agtggatcga tgtatccata gatgcgtgaa 2520 taatatttca tgtagagaag gagaacaaat tagatcatgt agggttatca 2570 SEQ ID NO:42 SEQ ID NO:43 atgaatttga gtttgtgtat agcatctcca ctattgacca aatctaatag accagctgct 60 ttatcagcaa ttcatacagc tagtacatcc catggtggcc aaaccaaccc tacgaatctg 120 ataatcgata cgaccaagga gagaatacaa aaacaattca aaaatgttga aatttcagtt 180 tcttcttatg atactgcgtg ggttgccatg gttccatcac ctaattctcc aaagtctcca 240 tgtttcccag aatgtttgaa ttggctgatt aacaaccagt tgaatgatgg atcttggggt 300 ttagtcaatc acacgcacaa tcacaaccat ccacttttga aagattcttt atcctcaact 360 ttggcttgca tcgtggccct aaagagatgg aacgtaggtg aggatcagat taacaagggg 420 cttagtttca ttgaatctaa cttggcttcc gcgactgaaa aatctcaacc atctccaata 480 ggattcgata tcatctttcc aggtctgtta gagtacgcca aaaatctaga tatcaactta 540 ctgtctaagc aaactgattt ctcactaatg ttacacaaga gagaattaga acaaaagaga 600 tgtcattcaa acgaaatgga tggttaccta gcttatatct ctgaaggtct tggtaatctt 660 tacgattgga atatggtgaa aaagtaccag atgaaaaatg gctcagtttt caattcccct 720 tctgcaactg cggcagcatt cattaaccat caaaatccag gatgcctgaa ctatttgaat 780 tcactactag acaaattcgg caacgcagtt ccaactgtat accctcacga tttgtttatc 840 agattgagta tggtggatac aattgaaaga cttggtatat cccaccactt tagagtcgag 900 atcaaaaatg ttttggatga gacataccgt tgttgggtgg agagagatga acaaatcttt 960 atggatgttg tgacgtgcgc gttggccttt agattgttgc gtattaacgg ttacgaagtt 1020 agtccagatc cacttgccga aattacaaac gaattagctt taaaggatga atacgccgct 1080 cttgaaacat atcatgcgtc acatatcctt taccaagagg acttatcatc tggaaaacaa 1140 attcttaaat ctgctgattt cctgaaggaa atcatatcca ctgatagtaa tagactgtcc 1200 aaactgatcc ataaagaggt tgaaaatgca cttaagttcc ctattaacac cggcttagaa 1260 cgtattaaca caagacgtaa catccagctt tacaacgtag acaatactag aatcttgaaa 1320 CA ()2973674 2017-07-12 accacttacc attcttccaa catatcaaac actgattacc taagattagc tgttgaagat 1380 ttctacacat gtcagtctat ctatagagaa gagctgaaag gattagagag atgggtcgtt 1440 gagaataagc tagatcaatt gaaatttgcc agacaaaaga cagcttattg ttacttctca 1500 gttgccgcca ctttatcaag tccagaattg tcagatgcac gtatttcttg ggctaaaaac 1560 ggaattttga caactgttgt tgatgatttc tttgatattg gcgggacaat cgacgaattg 1620 acaaacctga ttcaatgcgt tgaaaagtgg aatgtcgatg tcgataaaga ctgttgctca 1680 gaacatgtta gaatactgtt cttggctctg aaagatgcta tctgttggat cggggatgag 1740 gctttcaaat ggcaagctag agatgtgacg tctcacgtca ttcaaacctg gctagaactg 1800 atgaactcta tgttgagaga agcaatttgg actagagatg catacgttcc tacattaaac 1860 gagtatatgg aaaacgctta tgtctccttt gctttgggtc ctatcgttaa gcctgccata 1920 tactttgtag gaccaaagct atccgaggaa atcgtcgaat catcagaata ccataacttg 1980 ttcaagttaa tgtccacaca aggcagatta cttaatgata ttcattcttt caaaagagag 2040 tttaaggaag gaaagttaaa tgctgttgct ctgcatcttt ctaatggcga aagtggtaaa 2100 gtcgaagagg aagtagttga ggaaatgatg atgatgatca aaaacaagag aaaggagttg 2160 atgaaactaa tcttcgaaga gaacggttca attgttccta gagcatgtaa ggatgcattt 2220 tggaacatgt gtcatgtgct aaactttttc tacgcaaacg acgatggttt tactgggaac 2280 acaatactag atacagtaaa agacatcata tacaaccctt tggtcttagt aaacgaaaac 2340 gaggagcaaa gataa 2355 SEQ ID NO:44 SEQ ID NO:45 atgaatctgt ccctttgtat agctagtcca ctgttgacaa aatcttctag accaactgct 60 ctttctgcaa ttcatactgc cagtactagt catggaggtc aaacaaaccc aacaaatttg 120 ataatcgata ctactaagga gagaatccaa aagctattca aaaatgttga aatctcagta 180 tcatcttatg acaccgcatg ggttgcaatg gtgccatcac ctaattcccc aaaaagtcca 240 tgttttccag agtgcttgaa ttggttaatc aataatcagt taaacgatgg ttcttggggt 300 ttagtcaacc acactcataa ccacaatcat ccattattga aggactcttt atcatcaaca 360 ttagcctgta ttgttgcatt gaaaagatgg aatgtaggtg aagatcaaat caacaagggt 420 ttatcattca tagaatccaa tctagcttct gctaccgaca aatcacaacc atctccaatc 480 gggttcgaca taatcttccc tggtttgctg gagtatgcca aaaaccttga tatcaactta 540 ctgtctaaac aaacagattt ctctttgatg ctacacaaaa gagagttaga gcagaaaaga 600 tgccattcta acgaaattga cgggtactta gcatatatct cagaaggttt gggtaatttg 660 tatgactgga acatggtcaa aaagtatcag atgaaaaatg gatccgtatt caattctcct 720 tctgcaactg ccgcagcatt cattaatcat caaaaccctg ggtgtcttaa ctacttgaac 780 tcactattag ataagtttgg aaatgcagtt ccaacagtct atcctttgga cttgtacatc 840 agattatcta tggttgacac tatagagaga ttaggtattt ctcatcattt cagagttgag 900 atcaaaaatg ttttggacga gacatacaga tgttgggtcg aaagagatga gcaaatcttt 960 atggatgtcg tgacctgcgc tctggctttt agattgctaa ggatacacgg atacaaagta 1020 tctcctgatc aactggctga gattacaaac gaactggctt tcaaagacga atacgccgca 1080 ttagaaacat accatgcatc ccaaatactt taccaggaag acctaagttc aggaaaacaa 1140 atcttgaagt ctgcagattt cctgaaaggc attctgtcta cagatagtaa taggttgtct 1200 aaattgatac acaaggaagt agaaaacgca ctaaagtttc ctattaacac tggtttagag 1260 CA ()2973674 2017-07-12 agaatcaata ctaggagaaa cattcagctg tacaacgtag ataatacaag gattcttaag 1320 accacctacc atagttcaaa catttccaac acctattact taagattagc tgtcgaagac 1380 ttttacactt gtcaatcaat ctacagagag gagttaaagg gcctagaaag atgggtagtt 1440 caaaacaagt tggatcaact gaagtttgct agacagaaga cagcatactg ttatttctct 1500 gttgctgcta ccctttcatc cccagaattg tctgatgcca gaataagttg ggccaaaaat 1560 ggtattctta caactgtagt cgatgatttc tttgatattg gaggtactat tgatgaactg 1620 acaaatctta ttcaatgtgt tgaaaagtgg aacgtggatg tagataagga ttgctgcagt 1680 gaacatgtga gaatactttt cctggctcta aaagatgcaa tatgttggat tggcgacgag 1740 gccttcaagt ggcaagctag agatgttaca tctcatgtca tccaaacttg gcttgaactg 1800 atgaactcaa tgctaagaga agcaatctgg acaagagatg catacgttcc aacattgaac 1860 gaatacatgg aaaacgctta cgtctcattt gccttgggtc ctattgttaa gccagccata 1920 tactttgttg ggccaaagtt atccgaagag attgttgagt cttccgaata tcataaccta 1980 ttcaagttaa tgtcaacaca aggcagactt ctgaacgata tccactcctt caaaagagaa 2040 ttcaaggaag gtaagctaaa cgctgttgct ttgcacttgt ctaatggtga atctggcaaa 2100 gtggaagagg aagtcgttga ggaaatgatg atgatgatca aaaacaagag aaaggaattg 2160 atgaaattga ttttcgagga aaatggttca atcgtaccta gagcttgtaa agatgctttt 2220 tggaatatgt gccatgttct taacttcttt tacgctaatg atgatggctt cactggaaat 2280 acaatattgg atacagttaa agatatcatc tacaacccac ttgttttggt caatgagaac 2340 gaggaacaaa gataa 2355 SEQ ID NO:46 SEQ ID NO:47 atggctatgc cagtgaagct aacacctgcg tcattatcct taaaagctgt gtgctgcaga 60 ttctcatccg gtggccatgc tttgagattc gggagtagtc tgccatgttg gagaaggacc 120 cctacccaaa gatctacttc ttcctctact actagaccag ctgccgaagt gtcatcaggt 180 aagagtaaac aacatgatca ggaagctagt gaagcgacta tcagacaaca attacaactt 240 gtggatgtcc tggagaatat gggaatatcc agacattttg ctgcagagat aaagtgcata 300 ctagacagaa cttacagatc ttggttacaa agacacgagg aaatcatgct ggacactatg 360 acatgtgcta tggcttttag aatcctaaga ttgaacggat acaacgtttc atcagatgaa 420 ctataccacg ttgtagaggc atctggtctg cataattctt tgggtgggta tcttaacgat 480 accagaacac tacttgaatt acacaaggct tcaacagtta gtatctctga ggatgaatct 540 atcttagatt caattggctc tagatccaga acattgctta gagaacaatt ggagtctggt 600 ggcgcactga gaaagccttc tttattcaaa gaggttgaac atgcactgga tggacctttt 660 tacaccacac ttgatagact tcatcatagg tggaatattg aaaacttcaa cattattgag 720 caacacatgt tggagactcc atacttatct aaccagcata catcaaggga tatcctagca 780 ttgtcaatta gagatttttc ctcctcacaa ttcacttatc aacaagagct acagcatctg 840 gagagttggg ttaaggaatg tagattagat caactacagt tcgcaagaca gaaattagcg 900 tacttttacc tatcagccgc aggcaccatg ttttctcctg agctttctga tgcgagaaca 960 ttatgggcca aaaacggggt gttgacaact attgttgatg atttctttga tgttgccggt 1020 tctaaagagg aattggaaaa cttagtcatg ctggtcgaaa tgtgggatga acatcacaaa 1080 gttgaattct attctgagca ggtcgaaatc atcttctctt ccatctacga ttctgtcaac 1140 caattgggtg agaaggcctc tttggttcaa gacagatcaa ttacaaaaca ccttgttgaa 1200 CA ()2973674 2017-07-12 atatggttag acttgttaaa gtccatgatg acggaagttg aatggagact gtcaaaatac 1260 gtgcctacag aaaaggaata catgattaat gcctctctta tcttcggcct aggtccaatc 1320 gttttaccag ctttgtattt cgttggtcca aagatttcag aaagtatagt aaaggaccca 1380 gaatatgatg aattgttcaa actaatgtca acatgtggta gattgttgaa tgacgtgcaa 1440 acgttcgaaa gagaatacaa tgagggtaaa ctgaattctg tcagtctatt ggttcttcac 1500 ggaggcccaa tgtctatttc agacgcaaag aggaaattac aaaagcctat tgatacgtgt 1560 agaagagatc ttctttcttt ggtccttaga gaagagtctg tagtaccaag accatgtaag 1620 gaactattct ggaaaatgtg taaagtgtgc tatttctttt actcaacaac tgatgggttt 1680 tctagtcaag tcgaaagagc aaaagaggta gacgctgtca taaatgagcc actgaagttg 1740 caaggttctc atacactggt atctgatgtt taa 1773 SEQ ID NO:48 SEQ ID NO:49 atgcagaact tccatggtac aaaggaaagg atcaaaaaga tgtttgacaa gattgaattg 60 tccgtttctt cttatgatac agcctgggtt gcaatggtcc catcccctga ttgcccagaa 120 acaccttgtt ttccagaatg tactaaatgg atcctagaaa atcagttggg tgatggtagt 180 tggtcacttc ctcatggcaa tccacttcta gttaaagatg cattatcttc cactcttgct 240 tgtattctgg ctcttaaaag atggggaatc ggtgaggaac agattaacaa aggactgaga 300 ttcatagaac tcaactctgc tagtgtaacc gataacgaac aacacaaacc aattggattt 360 gacattatct ttccaggtat gattgaatac gctatagact tagacctgaa tctaccacta 420 aaaccaactg acattaactc catgttgcat cgtagagccc ttgaattgac atcaggtgga 480 ggcaaaaatc tagaaggtag aagagcttac ttggcctacg tctctgaagg aatcggtaag 540 ctgcaagatt gggaaatggc tatgaaatac caacgtaaaa acggatctct gttcaatagt 600 ccatcaacaa ctgcagctgc attcatccat atacaagatg ctgaatgcct ccactatatt 660 cgttctcttc tccagaaatt tggaaacgca gtccctacaa tataccctct cgatatctat 720 gccagacttt caatggtaga tgccctggaa cgtcttggta ttgatagaca tttcagaaag 780 gagagaaagt tcgttctgga tgaaacatac agattttggt tgcaaggaga agaggagatt 840 ttctccgata acgcaacctg tgctttggcc ttcagaatat tgagacttaa tggttacgat 900 gtctctcttg aagatcactt ctctaactct ctgggcggtt acttaaagga ctcaggagca 960 gctttagaac tgtacagagc cctccaattg tcttacccag acgagtccct cctggaaaag 1020 caaaattcta gaacttctta cttcttaaaa caaggtttat ccaatgtctc cctctgtggt 1080 gacagattgc gtaaaaacat aattggagag gtgcatgatg ctttaaactt ttccgaccac 1140 gctaacttac aaagattagc tattcgtaga aggattaagc attacgctac tgacgataca 1200 aggattctaa aaacttccta cagatgctca acaatcggta accaagattt tctaaaactt 1260 gcagtggaag atttcaatat ctgtcaatca atacaaagag aggaattcaa gcatattgaa 1320 agatgggtcg ttgaaagacg tctagacaag ttaaagttcg ctagacaaaa agaggcctat 1380 tgctatttct cagccgcagc aacattgttt gcccctgaat tgtctgatgc tagaatgtct 1440 tgggccaaaa atggtgtatt gacaactgtg gttgatgatt tcttcgatgt cggaggctct 1500 gaagaggaat tagttaactt gatagaattg atcgagcgtt gggatgtgaa tggcagtgca 1560 gatttttgta gtgaggaagt tgagattatc tattctgcta tccactcaac tatctctgaa 1620 ataggtgata agtcatttgg ctggcaaggt agagatgtaa agtctcaagt tatcaagatc 1680 tggctggact tattgaaatc aatgttaact gaagctcaat ggtcttcaaa caagtctgtt 1740 cctaccctag atgagtatat gacaaccgcc catgtttcat tcgcacttgg tccaattgta 1800 cttccagcct tatacttcgt tggcccaaag ttgtcagaag aggttgcagg tcatcctgaa 1860 ctactaaacc tctacaaagt cacatctact tgtggcagac tactgaatga ttggagaagt 1920 tttaagagag aatccgagga aggtaagctc aacgctatta gtttatacat gatccactcc 1980 CA ()2973674 2017-07-12 ggtggtgctt ctacagaaga ggaaacaatc gaacatttca aaggtttgat tgattctcag 2040 agaaggcaac tgttacaatt ggtgttgcaa gagaaggata gtatcatacc tagaccatgt 2100 aaagatctat tttggaatat gattaagtta ttacacactt tctacatgaa agatgatggc 2160 ttcacctcaa atgagatgag gaatgtagtt aaggcaatca ttaacgaacc aatctcactg 2220 gatgaattat ga 2232 SEQ ID NO:50 SEQ ID NO:51 atgtctatca accttcgctc ctccggttgt tcgtctccga tctcagctac tttggaacga 60 ggattggact cagaagtaca gacaagagct aacaatgtga gctttgagca aacaaaggag 120 aagattagga agatgttgga gaaagtggag ctttctgttt cggcctacga tactagttgg 180 gtagcaatgg ttccatcacc gagctcccaa aatgctccac ttttcccaca gtgtgtgaaa 240 tggttattgg ataatcaaca tgaagatgga tcttggggac ttgataacca tgaccatcaa 300 tctcttaaga aggatgtgtt atcatctaca ctggctagta tcctcgcgtt aaagaagtgg 360 ggaattggtg aaagacaaat aaacaagggt ctccagttta ttgagctgaa ttctgcatta 420 gtcactgatg aaaccataca gaaaccaaca gggtttgata ttatatttcc tgggatgatt 480 aaatatgcta gagatttgaa tctgacgatt ccattgggct cagaagtggt ggatgacatg 540 atacgaaaaa gagatctgga tcttaaatgt gatagtgaaa agttttcaaa gggaagagaa 600 gcatatctgg cctatgtttt agaggggaca agaaacctaa aagattggga tttgatagtc 660 aaatatcaaa ggaaaaatgg gtcactgttt gattctccag ccacaacagc agctgctttt 720 actcagtttg ggaatgatgg ttgtctccgt tatctctgtt ctctccttca gaaattcgag 780 gctgcagttc cttcagttta tccatttgat caatatgcac gccttagtat aattgtcact 840 cttgaaagct taggaattga tagagatttc aaaaccgaaa tcaaaagcat attggatgaa 900 acctatagat attggcttcg tggggatgaa gaaatatgtt tggacttggc cacttgtgct 960 ttggctttcc gattattgct tgctcatggc tatgatgtgt cttacgatcc gctaaaacca 1020 tttgcagaag aatctggttt ctctgatact ttggaaggat atgttaagaa tacgttttct 1080 gtgttagaat tatttaaggc tgctcaaagt tatccacatg aatcagcttt gaagaagcag 1140 tgttgttgga ctaaacaata tctggagatg gaattgtcca gctgggttaa gacctctgtt 1200 cgagataaat acctcaagaa agaggtcgag gatgctcttg cttttccctc ctatgcaagc 1260 ctagaaagat cagatcacag gagaaaaata ctcaatggtt ctgctgtgga aaacaccaga 1320 gttacaaaaa cctcatatcg tttgcacaat atttgcacct ctgatatcct gaagttagct 1380 gtggatgact tcaatttctg ccagtccata caccgtgaag aaatggaacg tcttgatagg 1440 tggattgtgg agaatagatt gcaggaactg aaatttgcca gacagaagct ggcttactgt 1500 tatttctctg gggctgcaac tttattttct ccagaactat ctgatgctcg tatatcgtgg 1560 gccaaaggtg gagtacttac aacggttgta gacgacttct ttgatgttgg agggtccaaa 1620 gaagaactgg aaaacctcat acacttggtc gaaaagtggg atttgaacgg tgttcctgag 1680 tacagctcag aacatgttga gatcatattc tcagttctaa gggacaccat tctcgaaaca 1740 ggagacaaag cattcaccta tcaaggacgc aatgtgacac accacattgt gaaaatttgg 1800 ttggatctgc tcaagtctat gttgagagaa gccgagtggt ccagtgacaa gtcaacacca 1860 agcttggagg attacatgga aaatgcgtac atatcatttg cattaggacc aattgtcctc 1920 ccagctacct atctgatcgg acctccactt ccagagaaga cagtcgatag ccaccaatat 1980 aatcagctct acaagctcgt gagcactatg ggtcgtcttc taaatgacat acaaggtttt 2040 aagagagaaa gcgcggaagg gaagctgaat gcggtttcat tgcacatgaa acacgagaga 2100 CA ()2973674 2017-07-12 gacaatcgca gcaaagaagt gatcatagaa tcgatgaaag gtttagcaga gagaaagagg 2160 gaagaattgc ataagctagt tttggaggag aaaggaagtg tggttccaag ggaatgcaaa 2220 gaagcgttct tgaaaatgag caaagtgttg aacttatttt acaggaagga cgatggattc 2280 acatcaaatg atctgatgag tcttgttaaa tcagtgatct acgagcctgt tagcttacag 2340 aaagaatctt taacttga 2358 SEQ ID NO:52 SEQ ID NO:53 atggaatttg atgaaccatt ggttgacgaa gcaagatctt tagtgcagcg tactttacaa 60 gattatgatg acagatacgg cttcggtact atgtcatgtg ctgcttatga tacagcctgg 120 gtgtctttag ttacaaaaac agtcgatggg agaaaacaat ggcttttccc agagtgtttt 180 gaatttctac tagaaacaca atctgatgcc ggaggatggg aaatcgggaa ttcagcacca 240 atcgacggta tattgaatac agctgcatcc ttacttgctc taaaacgtca cgttcaaact 300 gagcaaatca tccaacctca acatgaccat aaggatctag caggtagagc tgaacgtgcc 360 gctgcatctt tgagagcaca attggctgca ttggatgtgt ctacaactga acacgtcggt 420 tttgagataa ttgttcctgc aatgctagac ccattagaag ccgaagatcc atctctagtt 480 ttcgattttc cagctaggaa acctttgatg aagattcatg atgctaagat gagtagattc 540 aggccagaat acttgtatgg caaacaacca atgaccgcct tacattcatt agaggctttc 600 ataggcaaaa tcgacttcga taaggtaaga caccaccgta cccatgggtc tatgatgggt 660 tctccttcat ctaccgcagc ctacttaatg cacgcttcac aatgggatgg tgactcagag 720 gcttacctta gacacgtgat taaacacgca gcagggcagg gaactggtgc tgtaccatct 780 gctttcccat caacacattt tgagtcatct tggattctta ccacattgtt tagagctgga 840 ttttcagctt ctcatcttgc ctgtgatgag ttgaacaagt tggtcgagat acttgagggc 900 tcattcgaga aggaaggtgg ggcaatcggt tacgctccag ggtttcaagc agatgttgat 960 gatactgcta aaacaataag tacattagca gtccttggaa gagatgctac accaagacaa 1020 atgatcaagg tatttgaagc taatacacat tttagaacat accctggtga aagagatcct 1080 tctttgacag ctaattgtaa tgctctatca gccttactac accaaccaga tgcagcaatg 1140 tatggatctc aaattcaaaa gattaccaaa tttgtctgtg actattggtg gaagtctgat 1200 ggtaagatta aagataagtg gaacacttgc tacttgtacc catctgtctt attagttgag 1260 gttttggttg atcttgttag tttattggag cagggtaaat tgcctgatgt tttggatcaa 1320 gagcttcaat acagagtcgc catcacattg ttccaagcat gtttaaggcc attactagac 1380 caagatgccg aaggatcatg gaacaagtct atcgaagcca cagcctacgg catccttatc 1440 ctaactgaag ctaggagagt ttgtttcttc gacagattgt ctgagccatt gaatgaggca 1500 atccgtagag gtatcgcttt cgccgactct atgtctggaa ctgaagctca gttgaactac 1560 atttggatcg aaaaggttag ttacgcacct gcattattga ctaaatccta tttgttagca 1620 gcaagatggg ctgctaagtc tcctttaggc gcttccgtag gctcttcttt gtggactcca 1680 ccaagagaag gattggataa gcatgtcaga ttattccatc aagctgagtt attcagatcc 1740 cttccagaat gggaattaag agcctccatg attgaagcag ctttgttcac accacttcta 1800 agagcacata gactagacgt tttccctaga caagatgtag gtgaagacaa atatcttgat 1860 gtagttccat tcttttggac tgccgctaac aacagagata gaacttacgc ttccactcta 1920 ttcctttacg atatgtgttt tatcgcaatg ttaaacttcc agttagacga attcatggag 1980 gccacagccg gtatcttatt cagagatcat atggatgatt tgaggcaatt gattcatgat 2040 CA ()2973674 2017-07-12 cttttggcag agaaaacttc cccaaagagt tctggtagaa gtagtcaggg cacaaaagat 2100 gctgactcag gtatagagga agacgtgtca atgtccgatt cagcttcaga ttcccaggat 2160 agaagtccag aatacgactt ggttttcagt gcattgagta cctttacaaa acatgtcttg 2220 caacacccat ctatacaaag tgcctctgta tgggatagaa aactacttgc tagagagatg 2280 aaggcttact tacttgctca tatccaacaa gcagaagatt caactccatt gtctgaattg 2340 aaagatgtgc ctcaaaagac tgatgtaaca agagtttcta catctactac taccttcttt 2400 aactgggtta gaacaacttc cgcagaccat atatcctgcc catactcctt ccactttgta 2460 gcatgccatc taggcgcagc attgtcacct aaagggtcta acggtgattg ctatccttca 2520 gctggtgaga agttcttggc agctgcagtc tgcagacatt tggccaccat gtgtagaatg 2580 tacaacgatc ttggatcagc tgaacgtgat tctgatgaag gtaatttgaa ctccttggac 2640 ttccctgaat tcgccgattc cgcaggaaac ggagggatag aaattcagaa ggccgctcta 2700 ttaaggttag ctgagtttga gagagattca tacttagagg ccttccgtcg tttacaagat 2760 gaatccaata gagttcacgg tccagccggt ggtgatgaag ccagattgtc cagaaggaga 2820 atggcaatcc ttgaattctt cgcccagcag gtagatttgt acggtcaagt atacgtcatt 2880 agggatattt ccgctcgtat tcctaaaaac gaggttgaga aaaagagaaa attggatgat 2940 gctttcaatt ga 2952 SEQ ID NO:54 SEQ ID NO:55 atggcttcta gtacacttat ccaaaacaga tcatgtggcg tcacatcatc tatgtcaagt 60 tttcaaatct tcagaggtca accactaaga tttcctggca ctagaacccc agctgcagtt 120 caatgcttga aaaagaggag atgccttagg ccaaccgaat ccgtactaga atcatctcct 180 ggctctggtt catatagaat agtaactggc ccttctggaa ttaaccctag ttctaacggg 240 cacttgcaag agggttcctt gactcacagg ttaccaatac caatggaaaa atctatcgat 300 aacttccaat ctactctata tgtgtcagat atttggtctg aaacactaca gagaactgaa 360 tgtttgctac aagtaactga aaacgtccag atgaatgagt ggattgagga aattagaatg 420 tactttagaa atatgacttt aggtgaaatt tccatgtccc cttacgacac tgcttgggtg 480 gctagagttc cagcgttgga cggttctcat gggcctcaat tccacagatc tttgcaatgg 540 attatcgaca accaattacc agatggggac tggggcgaac cttctctttt cttgggttac 600 gatagagttt gtaatacttt agcctgtgtg attgcgttga aaacatgggg tgttggggca 660 caaaacgttg aaagaggaat tcagttccta caatctaaca tatacaagat ggaggaagat 720 gacgctaatc atatgccaat aggattcgaa atcgtattcc ctgctatgat ggaagatgcc 780 aaagcattag gtttggattt gccatacgat gctactattt tgcaacagat ttcagccgaa 840 agagagaaaa agatgaaaaa gatcccaatg gcaatggtgt acaaataccc aaccacttta 900 cttcactcct tagaaggctt gcatagagaa gttgattgga ataagttgtt acaattacaa 960 tctgaaaatg gtagttttct ttattcacct gcttcaaccg catgcgcctt aatgtacact 1020 aaggacgtta aatgttttga ttacttaaac cagttgttga tcaagttcga ccacgcatgc 1080 ccaaatgtat atccagtcga tctattcgaa agattatgga tggttgacag attgcagaga 1140 ttagggatct ccagatactt tgaaagagag attagagatt gtttacaata cgtctacaga 1200 CA ()2973674 2017-07-12 tattggaaag attgtggaat cggatgggct tctaactctt ccgtacaaga tgttgatgat 1260 acagccatgg cgtttagact tttaaggact catggtttcg acgtaaagga agattgcttt 1320 agacagtttt tcaaggacgg agaattcttc tgcttcgcag gccaatcatc tcaagcagtt 1380 acaggcatgt ttaatctttc aagagccagt caaacattgt ttccaggaga atctttattg 1440 aaaaaggcta gaaccttctc tagaaacttc ttgagaacaa agcatgagaa caacgaatgt 1500 ttcgataaat ggatcattac taaagatttg gctggtgaag tcgagtataa cttgaccttc 1560 ccatggtatg cctctttgcc tagattagaa cataggacat acttagatca atatggaatc 1620 gatgatatct ggataggcaa atctttatac aaaatgcctg ctgttaccaa cgaagttttc 1680 ctaaagttgg caaaggcaga ctttaacatg tgtcaagctc tacacaaaaa ggaattggaa 1740 caagtgataa agtggaacgc gtcctgtcaa ttcagagatc ttgaattcgc cagacaaaaa 1800 tcagtagaat gctattttgc tggtgcagcc acaatgttcg aaccagaaat ggttcaagct 1860 agattagtct gggcaagatg ttgtgtattg acaactgtct tagacgatta ctttgaccac 1920 gggacacctg ttgaggaact tagagtgttt gttcaagctg tcagaacatg gaatccagag 1980 ttgatcaacg gtttgccaga gcaagctaaa atcttgttta tgggcttata caaaacagtt 2040 aacacaattg cagaggaagc attcatggca cagaaaagag acgtccatca tcatttgaaa 2100 cactattggg acaagttgat aacaagtgcc ctaaaggagg ccgaatgggc agagtcaggt 2160 tacgtcccaa catttgatga atacatggaa gtagctgaaa tttctgttgc tctagaacca 2220 attgtctgta gtaccttgtt ctttgcgggt catagactag atgaggatgt tctagatagt 2280 tacgattacc atctagttat gcatttggta aacagagtcg gtagaatctt gaatgatata 2340 caaggcatga agagggaggc ttcacaaggt aagatctcat cagttcaaat ctacatggag 2400 gaacatccat ctgttccatc tgaggccatg gcgatcgctc atcttcaaga gttagttgat 2460 aattcaatgc agcaattgac atacgaagtt cttaggttca ctgcggttcc aaaaagttgt 2520 aagagaatcc acttgaatat ggctaaaatc atgcatgcct tctacaagga tactgatgga 2580 ttctcatccc ttactgcaat gacaggattc gtcaaaaagg ttcttttcga acctgtgcct 2640 gagtaa 2646 SEQ ID NO:56 SEQ ID NO:57 atgcctggta aaattgaaaa tggtacccca aaggacctca agactggaaa tgattttgtt 60 tctgctgcta agagtttact agatcgagct ttcaaaagtc atcattccta ctacggatta 120 tgctcaactt catgtcaagt ttatgataca gcttgggttg caatgattcc aaaaacaaga 180 gataatgtaa aacagtggtt gtttccagaa tgtttccatt acctcttaaa aacacaagcc 240 gcagatggct catggggttc attgcctaca acacagacag cgggtatcct agatacagcc 300 tcagctgtgc tggcattatt gtgccacgca caagagcctt tacaaatatt ggatgtatct 360 ccagatgaaa tggggttgag aatagaacac ggtgtcacat ccttgaaacg tcaattagca 420 gtttggaatg atgtggagga caccaaccat attggcgtcg agtttatcat accagcctta 480 ctttccatgc tagaaaagga attagatgtt ccatcttttg aatttccatg taggtccatc 540 ttagagagaa tgcacgggga gaaattaggt catttcgacc tggaacaagt ttacggcaag 600 ccaagctcat tgttgcactc attggaagca tttctcggta agctagattt tgatcgacta 660 tcacatcacc tataccacgg cagtatgatg gcatctccat cttcaacggc tgcttatctt 720 attggggcta caaaatggga tgacgaagcc gaagattacc taagacatgt aatgcgtaat 780 CA ()2973674 2017-07-12 ggtgcaggac atgggaatgg aggtatttct ggtacatttc caactactca tttcgaatgt 840 agctggatta tagcaacgtt gttaaaggtt ggctttactt tgaagcaaat tgacggcgat 900 ggcttaagag gtttatcaac catcttactt gaggcgcttc gtgatgagaa tggtgtcata 960 ggctttgccc ctagaacagc agatgtagat gacacagcca aagctctatt ggccttgtca 1020 ttggtaaacc agccagtgtc acctgatatc atgattaagg tctttgaggg caaagaccat 1080 tttaccactt ttggttcaga aagagatcca tcattgactt ccaacctgca cgtcctttta 1140 tctttactta aacaatctaa cttgtctcaa taccatcctc aaatcctcaa aacaacatta 1200 ttcacttgta gatggtggtg gggttccgat cattgtgtca aagacaaatg gaatttgagt 1260 cacctatatc caactatgtt gttggttgaa gccttcactg aagtgctcca tctcattgac 1320 ggtggtgaat tgtctagtct gtttgatgaa tcctttaagt gtaagattgg tcttagcatc 1380 tttcaagcgg tacttagaat aatcctcacc caagacaacg acggctcttg gagaggatac 1440 agagaacaga cgtgttacgc aatattggct ttagttcaag cgagacatgt atgctttttc 1500 actcacatgg ttgacagact gcaatcatgt gttgatcgag gtttctcatg gttgaaatct 1560 tgctcttttc attctcaaga cctgacttgg acctctaaaa cagcttatga agtgggtttc 1620 gtagctgaag catataaact agctgcttta caatctgctt ccctggaggt tcctgctgcc 1680 accattggac attctgtcac gtctgccgtt ccatcaagtg atcttgaaaa atacatgaga 1740 ttggtgagaa aaactgcgtt attctctcca ctggatgagt ggggtctaat ggcttctatc 1800 atcgaatctt catttttcgt accattactg caggcacaaa gagttgaaat ataccctaga 1860 gataatatca aggtggacga agataagtac ttgtctatta tcccattcac atgggtcgga 1920 tgcaataata ggtctagaac tttcgcaagt aacagatggc tatacgatat gatgtacctt 1980 tcattactcg gctatcaaac cgacgagtac atggaagctg tagctgggcc agtgtttggg 2040 gatgtttcct tgttacatca aacaattgat aaggtgattg ataatacaat gggtaacctt 2100 gcgagagcca atggaacagt acacagtggt aatggacatc agcacgaatc tcctaatata 2160 ggtcaagtcg aggacacctt gactcgtttc acaaattcag tcttgaatca caaagacgtc 2220 cttaactcta gctcatctga tcaagatact ttgagaagag agtttagaac attcatgcac 2280 gctcatataa cacaaatcga agataactca cgattcagta agcaagcctc atccgatgcg 2340 ttttcctctc ctgaacaatc ttactttcaa tgggtgaact caactggtgg ctcacatgtc 2400 gcttgcgcct attcatttgc cttctctaat tgcctcatgt ctgcaaattt gttgcagggt 2460 aaagacgcat ttccaagcgg aacgcaaaag tacttaatct cctctgttat gagacatgcc 2520 acaaacatgt gtagaatgta taacgacttt ggctctattg ccagagacaa cgctgagaga 2580 aatgttaata gtattcattt tcctgagttt actctctgta acggaacttc tcaaaaccta 2640 gatgaaagga aggaaagact tctgaaaatc gcaacttacg aacaagggta tttggataga 2700 gcactagagg ccttggaaag acagagtaga gatgatgccg gagacagagc tggatctaaa 2760 gatatgagaa agttgaaaat cgttaagtta ttctgtgatg ttacggactt atacgatcag 2820 ctctacgtta tcaaagattt gtcatcctct atgaagtaa 2859 SEQ ID NO:58 SEQ ID NO:59 atggatgctg tgacgggttt gttaactgtc ccagcaaccg ctataactat tggtggaact 60 gctgtagcat tggcggtagc gctaatcttt tggtacctga aatcctacac atcagctaga 120 CA ()2973674 2017-07-12 agatcccaat caaatcatct tccaagagtg cctgaagtcc caggtgttcc attgttagga 180 aatctgttac aattgaagga gaaaaagcca tacatgactt ttacgagatg ggcagcgaca 240 tatggaccta tctatagtat caaaactggg gctacaagta tggttgtggt atcatctaat 300 gagatagcca aggaggcatt ggtgaccaga ttccaatcca tatctacaag gaacttatct 360 aaagccctga aagtacttac agcagataag acaatggtcg caatgtcaga ttatgatgat 420 tatcataaaa cagttaagag acacatactg accgccgtct tgggtcctaa tgcacagaaa 480 aagcatagaa ttcacagaga tatcatgatg gataacatat ctactcaact tcatgaattc 540 gtgaaaaaca acccagaaca ggaagaggta gaccttagaa aaatctttca atctgagtta 600 ttcggcttag ctatgagaca agccttagga aaggatgttg aaagtttgta cgttgaagac 660 ctgaaaatca ctatgaatag agacgaaatc tttcaagtcc ttgttgttga tccaatgatg 720 ggagcaatcg atgttgattg gagagacttc tttccatacc taaagtgggt cccaaacaaa 780 aagttcgaaa atactattca acaaatgtac atcagaagag aagctgttat gaaatcttta 840 atcaaagagc acaaaaagag aatagcgtca ggcgaaaagc taaatagtta tatcgattac 900 cttttatctg aagctcaaac tttaaccgat cagcaactat tgatgtcctt gtgggaacca 960 atcattgaat cttcagatac aacaatggtc acaacagaat gggcaatgta cgaattagct 1020 aaaaacccta aattgcaaga taggttgtac agagacatta agtccgtctg tggatctgaa 1080 aagataaccg aagagcatct atcacagctg ccttacatta cagctatttt ccacgaaaca 1140 ctgagaagac actcaccagt tcctatcatt cctctaagac atgtacatga agataccgtt 1200 ctaggcggct accatgttcc tgctggcaca gaacttgccg ttaacatcta cggttgcaac 1260 atggacaaaa acgtttggga aaatccagag gaatggaacc cagaaagatt catgaaagag 1320 aatgagacaa ttgattttca aaagacgatg gccttcggtg gtggtaagag agtttgtgct 1380 ggttccttgc aagccctttt aactgcatct attgggattg ggagaatggt tcaagagttc 1440 gaatggaaac tgaaggatat gactcaagag gaagtgaaca cgataggcct aactacacaa 1500 atgttaagac cattgagagc tattatcaaa cctaggatct aa 1542 SEQ ID NO:60 SEQ ID NO:61 aagcttacta gtaaaatgga cggtgtcatc gatatgcaaa ccattccatt gagaaccgct 60 attgctattg gtggtactgc tgttgctttg gttgttgcat tatacttttg gttcttgaga 120 tcctacgctt ccccatctca tcattctaat catttgccac cagtacctga agttccaggt 180 gttccagttt tgggtaattt gttgcaattg aaagaaaaaa agccttacat gaccttcacc 240 aagtgggctg aaatgtatgg tccaatctac tctattagaa ctggtgctac ttccatggtt 300 gttgtctctt ctaacgaaat cgccaaagaa gttgttgtta ccagattccc atctatctct 360 accagaaaat tgtcttacgc cttgaaggtt ttgaccgaag ataagtctat ggttgccatg 420 tctgattatc acgattacca taagaccgtc aagagacata ttttgactgc tgttttgggt 480 ccaaacgccc aaaaaaagtt tagagcacat agagacacca tgatggaaaa cgtttccaat 540 gaattgcatg ccttcttcga aaagaaccca aatcaagaag tcaacttgag aaagatcttc 600 caatcccaat tattcggttt ggctatgaag caagccttgg gtaaagatgt tgaatccatc 660 tacgttaagg atttggaaac caccatgaag agagaagaaa tcttcgaagt tttggttgtc 720 gatccaatga tgggtgctat tgaagttgat tggagagact ttttcccata cttgaaatgg 780 gttccaaaca agtccttcga aaacatcatc catagaatgt acactagaag agaagctgtt 840 atgaaggcct tgatccaaga acacaagaaa agaattgcct ccggtgaaaa cttgaactcc 900 tacattgatt acttgttgtc tgaagcccaa accttgaccg ataagcaatt attgatgtct 960 ttgtgggaac ctattatcga atcttctgat accactatgg ttactactga atgggctatg 1020 tacgaattgg ctaagaatcc aaacatgcaa gacagattat acgaagaaat ccaatccgtt 1080 tgcggttccg aaaagattac tgaagaaaac ttgtcccaat tgccatactt gtacgctgtt 1140 ttccaagaaa ctttgagaaa gcactgtcca gttcctatta tgccattgag atatgttcac 1200 CA ()2973674 2017-07-12 gaaaacaccg ttttgggtgg ttatcatgtt ccagctggta ctgaagttgc tattaacatc 1260 tacggttgca acatggataa gaaggtctgg gaaaatccag aagaatggaa tccagaaaga 1320 ttcttgtccg aaaaagaatc catggacttg tacaaaacta tggcttttgg tggtggtaaa 1380 agagtttgcg ctggttcttt acaagccatg gttatttctt gcattggtat cggtagattg 1440 gtccaagatt ttgaatggaa gttgaaggat gatgccgaag aagatgttaa cactttgggt 1500 ttgactaccc aaaagttgca tccattattg gccttgatta acccaagaaa gtaactcgag 1560 ccgcgg 1566 SEQ ID NO:62 SEQ ID NO:63 atggccaccc tccttgagca tttccaagct atgccctttg ccatccctat tgcactggct 60 gctctgtctt ggctgttcct cttttacatc aaagtttcat tcttttccaa caagagtgct 120 caggctaagc tccctcctgt gccagtggtt cctgggctgc cggtgattgg gaatttactg 180 caactcaagg agaagaaacc ctaccagact tttacaaggt gggctgagga gtatggacca 240 atctattcta tcaggactgg tgcttccacc atggtcgttc tcaataccac ccaagttgca 300 aaagaggcca tggtgaccag atatttatcc atctcaacca gaaagctatc aaacgcacta 360 aagattctta ctgctgataa atgtatggtt gcaataagtg actacaacga ttttcacaag 420 atgataaagc gatacatact ctcaaatgtt cttggaccta gtgctcagaa gcgtcaccgg 480 agcaacagag ataccttgag agctaatgtc tgcagccgat tgcattctca agtaaagaac 540 tctcctcgag aagctgtgaa tttcagaaga gtttttgagt gggaactctt tggaattgca 600 ttgaagcaag cctttggaaa ggacatagaa aagcccattt atgtggagga acttggcact 660 acactgtcaa gagatgagat ctttaaggtt ctagtgcttg acataatgga gggtgcaatt 720 gaggttgatt ggagagattt cttcccttac ctgagatgga ttccgaatac gcgcatggaa 780 acaaaaattc agcgactcta tttccgcagg aaagcagtga tgactgccct gatcaacgag 840 cagaagaagc gaattgcttc aggagaggaa atcaactgtt atatcgactt cttgcttaag 900 gaagggaaga cactgacaat ggaccaaata agtatgttgc tttgggagac ggttattgaa 960 acagcagata ctacaatggt aacgacagaa tgggctatgt atgaagttgc taaagactca 1020 aagcgtcagg atcgtctcta tcaggaaatc caaaaggttt gtggatcgga gatggttaca 1080 gaggaatact tgtcccaact gccgtacctg aatgcagttt tccatgaaac gctaaggaag 1140 cacagtccgg ctgcgttagt tcctttaaga tatgcacatg aagataccca actaggaggt 1200 tactacattc cagctggaac tgagattgct ataaacatat acgggtgtaa catggacaag 1260 catcaatggg aaagccctga ggaatggaaa ccggagagat ttttggaccc gaaatttgat 1320 cctatggatt tgtacaagac catggctttt ggggctggaa agagggtatg tgctggttct 1380 cttcaggcaa tgttaatagc gtgcccgacg attggtaggc tggtgcagga gtttgagtgg 1440 aagctgagag atggagaaga agaaaatgta gatactgttg ggctcaccac tcacaaacgc 1500 tatccaatgc atgcaatcct gaagccaaga agtta 1535 SEQ ID NO:64 atggctacct tgttggaaca ttttcaagct atgccattcg ctattccaat tgctttggct 60 gctttgtctt ggttgttttt gttctacatc aaggtttctt tcttctccaa caaatccgct 120 caagctaaat tgccaccagt tccagttgtt ccaggtttgc cagttattgg taatttgttg 180 caattgaaag aaaagaagcc ataccaaacc ttcactagat gggctgaaga atatggtcca 240 atctactcta ttagaactgg tgcttctact atggttgtct tgaacactac tcaagttgcc 300 aaagaagcta tggttaccag atacttgtct atctctacca gaaagttgtc caacgccttg 360 aaaattttga ccgctgataa gtgcatggtt gccatttctg attacaacga tttccacaag 420 atgatcaaga gatatatctt gtctaacgtt ttgggtccat ctgcccaaaa aagacataga 480 tctaacagag ataccttgag agccaacgtt tgttctagat tgcattccca agttaagaac 540 CA ()2973674 2017-07-12 tctccaagag aagctgtcaa ctttagaaga gttttcgaat gggaattatt cggtatcgct 600 ttgaaacaag ccttcggtaa ggatattgaa aagccaatct acgtcgaaga attgggtact 660 actttgtcca gagatgaaat cttcaaggtt ttggtcttgg acattatgga aggtgccatt 720 gaagttgatt ggagagattt tttcccatac ttgcgttgga ttccaaacac cagaatggaa 780 actaagatcc aaagattata ctttagaaga aaggccgtta tgaccgcctt gattaacgaa 840 caaaagaaaa gaattgcctc cggtgaagaa atcaactgct acatcgattt cttgttgaaa 900 gaaggtaaga ccttgaccat ggaccaaatc tctatgttgt tgtgggaaac cgttattgaa 960 actgctgata ccacaatggt tactactgaa tgggctatgt acgaagttgc taaggattct 1020 aaaagacaag acagattata ccaagaaatc caaaaggtct gcggttctga aatggttaca 1080 gaagaatact tgtcccaatt gccatacttg aatgctgttt tccacgaaac tttgagaaaa 1140 cattctccag ctgctttggt tccattgaga tatgctcatg aagatactca attgggtggt 1200 tattacattc cagccggtac tgaaattgcc attaacatct acggttgcaa catggacaaa 1260 caccaatggg aatctccaga agaatggaag ccagaaagat ttttggatcc taagtttgac 1320 ccaatggact tgtacaaaac tatggctttt ggtgctggta aaagagtttg cgctggttct 1380 ttacaagcta tgttgattgc ttgtccaacc atcggtagat tggttcaaga atttgaatgg 1440 aagttgagag atggtgaaga agaaaacgtt gatactgttg gtttgaccac ccataagaga 1500 tatccaatgc atgctatttt gaagccaaga tcttaa 1536 SEQ ID NO:65 aagcttacta gtaaaatggc ctccatcacc catttcttac aagattttca agctactcca 60 ttcgctactg cttttgctgt tggtggtgtt tctttgttga tattcttctt cttcatccgt 120 ggtttccact ctactaagaa aaacgaatat tacaagttgc caccagttcc agttgttcca 180 ggtttgccag ttgttggtaa tttgttgcaa ttgaaagaaa agaagccata caagactttc 240 ttgagatggg ctgaaattca tggtccaatc tactctatta gaactggtgc ttctaccatg 300 gttgttgtta actctactca tgttgccaaa gaagctatgg ttaccagatt ctcttcaatc 360 tctaccagaa agttgtccaa ggctttggaa ttattgacct ccaacaaatc tatggttgcc 420 acctctgatt acaacgaatt tcacaagatg gtcaagaagt acatcttggc cgaattattg 480 ggtgctaatg ctcaaaagag acacagaatt catagagaca ccttgatcga aaacgtcttg 540 aacaaattgc atgcccatac caagaattct ccattgcaag ctgttaactt cagaaagatc 600 ttcgaatctg aattattcgg tttggctatg aagcaagcct tgggttatga tgttgattcc 660 ttgttcgttg aagaattggg tactaccttg tccagagaag aaatctacaa cgttttggtc 720 agtgacatgt tgaagggtgc tattgaagtt gattggagag actttttccc atacttgaaa 780 tggatcccaa acaagtcctt cgaaatgaag attcaaagat tggcctctag aagacaagcc 840 gttatgaact ctattgtcaa agaacaaaag aagtccattg cctctggtaa gggtgaaaac 900 tgttacttga attacttgtt gtccgaagct aagactttga ccgaaaagca aatttccatt 960 ttggcctggg aaaccattat tgaaactgct gatacaactg ttgttaccac tgaatgggct 1020 atgtacgaat tggctaaaaa cccaaagcaa caagacagat tatacaacga aatccaaaac 1080 gtctgcggta ctgataagat taccgaagaa catttgtcca agttgcctta cttgtctgct 1140 gtttttcacg aaaccttgag aaagtattct ccatctccat tggttccatt gagatacgct 1200 catgaagata ctcaattggg tggttattat gttccagccg gtactgaaat tgctgttaat 1260 atctacggtt gcaacatgga caagaatcaa tgggaaactc cagaagaatg gaagccagaa 1320 agatttttgg acgaaaagta cgatccaatg gacatgtaca agactatgtc ttttggttcc 1380 ggtaaaagag tttgcgctgg ttctttacaa gctagtttga ttgcttgtac ctccatcggt 1440 agattggttc aagaatttga atggagattg aaagacggtg aagttgaaaa cgttgatacc 1500 ttgggtttga ctacccataa gttgtatcca atgcaagcta tcttgcaacc tagaaactga 1560 ctcgagccgc gg 1572 SEQ ID NO:66 CA ()2973674 2017-07-12 SEQ ID NO:67 atgatttcct tgttgttggg ttttgttgtc tcctccttct tgtttatctt cttcttgaaa 60 aaattgttgt tcttcttcag tcgtcacaaa atgtccgaag tttctagatt gccatctgtt 120 ccagttccag gttttccatt gattggtaac ttgttgcaat tgaaagaaaa gaagccacac 180 aagactttca ccaagtggtc tgaattatat ggtccaatct actctatcaa gatgggttcc 240 tcttctttga tcgtcttgaa ctctattgaa accgccaaag aagctatggt cagtagattc 300 tcttcaatct ctaccagaaa gttgtctaac gctttgactg ttttgacctg caacaaatct 360 atggttgcta cctctgatta cgatgacttt cataagttcg tcaagagatg cttgttgaac 420 ggtttgttgg gtgctaatgc tcaagaaaga aaaagacatt acagagatgc cttgatcgaa 480 aacgttacct ctaaattgca tgcccatacc agaaatcatc cacaagaacc agttaacttc 540 agagccattt tcgaacacga attattcggt gttgctttga aacaagcctt cggtaaagat 600 gtcgaatcca tctatgtaaa agaattgggt gtcaccttgt ccagagatga aattttcaag 660 gttttggtcc acgacatgat ggaaggtgct attgatgttg attggagaga tttcttccca 720 tacttgaaat ggatcccaaa caactctttc gaagccagaa ttcaacaaaa gcacaagaga 780 agattggctg ttatgaacgc cttgatccaa gacagattga atcaaaacga ttccgaatcc 840 gatgatgact gctacttgaa tttcttgatg tctgaagcta agaccttgac catggaacaa 900 attgctattt tggtttggga aaccattatc gaaactgctg ataccacttt ggttactact 960 gaatgggcta tgtacgaatt ggccaaacat caatctgttc aagatagatt attcaaagaa 1020 atccaatccg tctgcggtgg tgaaaagatc aaagaagaac aattgccaag attgccttac 1080 gtcaatggtg tttttcacga aaccttgaga aagtattctc cagctccatt ggttccaatt 1140 agatacgctc atgaagatac ccaaattggt ggttatcata ttccagccgg ttctgaaatt 1200 gccattaaca tctacggttg caacatggat aagaagagat gggaaagacc tgaagaatgg 1260 tggccagaaa gatttttgga agatagatac gaatcctccg acttgcataa gactatggct 1320 tttggtgctg gtaaaagagt ttgtgctggt gctttacaag ctagtttgat ggctggtatt 1380 gctatcggta gattggttca agaattcgaa tggaagttga gagatggtga agaagaaaac 1440 gttgatactt acggtttgac ctcccaaaag ttgtatccat tgatggccat tatcaaccca 1500 agaagatctt aa 1512 SEQ ID NO:68 SEQ ID NO:69 aagcttacta gtaaaatgga catgatgggt attgaagctg ttccatttgc tactgctgtt 60 gttttgggtg gtatttcctt ggttgttttg atcttcatca gaagattcgt ttccaacaga 120 aagagatccg ttgaaggttt gccaccagtt ccagatattc caggtttacc attgattggt 180 aacttgttgc aattgaaaga aaagaagcca cataagacct ttgctagatg ggctgaaact 240 tacggtccaa ttttctctat tagaactggt gcttctacca tgatcgtctt gaattcttct 300 gaagttgcca aagaagctat ggtcactaga ttctcttcaa tctctaccag aaagttgtcc 360 aacgccttga agattttgac cttcgataag tgtatggttg ccacctctga ttacaacgat 420 tttcacaaaa tggtcaaggg tttcatcttg agaaacgttt taggtgctcc agcccaaaaa 480 agacatagat gtcatagaga taccttgatc gaaaacatct ctaagtactt gcatgcccat 540 gttaagactt ctccattgga accagttgtc ttgaagaaga ttttcgaatc cgaaattttc 600 ggtttggctt tgaaacaagc cttgggtaag gatatcgaat ccatctatgt tgaagaattg 660 ggtactacct tgtccagaga agaaattttt gccgttttgg ttgttgatcc aatggctggt 720 gctattgaag ttgattggag agattttttc ccatacttgt cctggattcc aaacaagtct 780 atggaaatga agatccaaag aatggatttt agaagaggtg ctttgatgaa ggccttgatt 840 ggtgaacaaa agaaaagaat cggttccggt gaagaaaaga actcctacat tgatttcttg 900 ttgtctgaag ctaccacttt gaccgaaaag caaattgcta tgttgatctg ggaaaccatc 960 CA ()2973674 2017-07-12 atcgaaattt ccgatacaac tttggttacc tctgaatggg ctatgtacga attggctaaa 1020 gacccaaata gacaagaaat cttgtacaga gaaatccaca aggtttgcgg ttctaacaag 1080 ttgactgaag aaaacttgtc caagttgcca tacttgaact ctgttttcca cgaaaccttg 1140 agaaagtatt ctccagctcc aatggttcca gttagatatg ctcatgaaga tactcaattg 1200 ggtggttacc atattccagc tggttctcaa attgccatta acatctacgg ttgcaacatg 1260 aacaaaaagc aatgggaaaa tcctgaagaa tggaagccag aaagattctt ggacgaaaag 1320 tatgacttga tggacttgca taagactatg gcttttggtg gtggtaaaag agtttgtgct 1380 ggtgctttac aagcaatgtt gattgcttgc acttccatcg gtagattcgt tcaagaattt 1440 gaatggaagt tgatgggtgg tgaagaagaa aacgttgata ctgttgcttt gacctcccaa 1500 aaattgcatc caatgcaagc cattattaag gccagagaat gactcgagcc gcgg 1554 SEQ ID NO:70 SEQ ID NO:71 aagcttaaaa tgagtaagtc taatagtatg aattctacat cacacgaaac cctttttcaa 60 caattggtct tgggtttgga ccgtatgcca ttgatggatg ttcactggtt gatctacgtt 120 gctttcggcg catggttatg ttcttatgtg atacatgttt tatcatcttc ctctacagta 180 aaagtgccag ttgttggata caggtctgta ttcgaaccta catggttgct tagacttaga 240 ttcgtctggg aaggtggctc tatcataggt caagggtaca ataagtttaa agactctatt 300 ttccaagtta ggaaattggg aactgatatt gtcattatac cacctaacta tattgatgaa 360 gtgagaaaat tgtcacagga caagactaga tcagttgaac ctttcattaa tgattttgca 420 ggtcaataca caagaggcat ggttttcttg caatctgact tacaaaaccg tgttatacaa 480 caaagactaa ctccaaaatt ggtttccttg accaaggtca tgaaggaaga gttggattat 540 gctttaacaa aagagatgcc tgatatgaaa aatgacgaat gggtagaagt agatatcagt 600 agtataatgg tgagattgat ttccaggatc tccgccagag tctttctagg gcctgaacac 660 tgtcgtaacc aggaatggtt gactactaca gcagaatatt cagaatcact tttcattaca 720 gggtttatct taagagttgt acctcatatc ttaagaccat tcatcgcccc tctattacct 780 tcatacagga ctctacttag aaacgtttca agtggtagaa gagtcatcgg tgacatcata 840 agatctcagc aaggggatgg taacgaagat atactttcct ggatgagaga tgctgccaca 900 ggagaggaaa agcaaatcga taacattgct cagagaatgt taattctttc tttagcatca 960 atccacacta ctgcgatgac catgacacat gccatgtacg atctatgtgc ttgccctgag 1020 tacattgaac cattaagaga tgaagttaaa tctgttgttg gggcttctgg ctgggacaag 1080 acagcgttaa acagatttca taagttggac tccttcctaa aagagtcaca aagattcaac 1140 ccagtattct tattgacatt caatagaatc taccatcaat ctatgacctt atcagatggc 1200 actaacattc catctggaac acgtattgct gttccatcac acgcaatgtt gcaagattct 1260 gcacatgtcc caggtccaac cccacctact gaatttgatg gattcagata tagtaagata 1320 cgttctgata gtaactacgc acaaaagtac ctattctcca tgaccgattc ttcaaacatg 1380 gctttcggat acggcaagta tgcttgtcca ggtagatttt acgcgtctaa tgagatgaaa 1440 ctaacattag ccattttgtt gctacaattt gagttcaaac taccagatgg taaaggtcgt 1500 cctagaaata tcactatcga ttctgatatg attccagacc caagagctag actttgcgtc 1560 agaaaaagat cacttagaga tgaatgaccg cgg 1593 SEQ ID NO:72 CA ()2973674 2017-07-12 SEQ ID NO:73 aagcttaaaa tggaagatcc tactgtctta tatgcttgtc ttgccattgc agttgcaact 60 ttcgttgtta gatggtacag agatccattg agatccatcc caacagttgg tggttccgat 120 ttgcctattc tatcttacat cggcgcacta agatggacaa gacgtggcag agagatactt 180 caagagggat atgatggcta cagaggatct acattcaaaa tcgcgatgtt agaccgttgg 240 atcgtgatcg caaatggtcc taaactagct gatgaagtca gacgtagacc agatgaagag 300 ttaaacttta tggacggatt aggagcattc gtccaaacta agtacacctt aggtgaagct 360 attcataacg atccatacca tgtcgatatc ataagagaaa aactaacaag aggccttcca 420 gccgtgcttc ctgatgtcat tgaagagttg acacttgcgg ttagacagta cattccaaca 480 gaaggtgatg aatgggtgtc cgtaaactgt tcaaaggccg caagagatat tgttgctaga 540 gcttctaata gagtctttgt aggtttgcct gcttgcagaa accaaggtta cttagatttg 600 gcaatagact ttacattgtc tgttgtcaag gatagagcca tcatcaatat gtttccagaa 660 ttgttgaagc caatagttgg cagagttgta ggtaacgcca ccagaaatgt tcgtagagct 720 gttccttttg ttgctccatt ggtggaggaa agacgtagac ttatggaaga gtacggtgaa 780 gactggtctg aaaaacctaa tgatatgtta cagtggataa tggatgaagc tgcatccaga 840 gatagttcag tgaaggcaat cgcagagaga ttgttaatgg tgaacttcgc ggctattcat 900 acctcatcaa acactatcac tcatgctttg taccaccttg ccgaaatgcc tgaaactttg 960 caaccactta gagaagagat cgaaccatta gtcaaagagg agggctggac caaggctgct 1020 atgggaaaaa tgtggtggtt agattcattt ctaagagaat ctcaaagata caatggcatt 1080 aacatcgtat ctttaactag aatggctgac aaagatatta cattgagtga tggcacattt 1140 ttgccaaaag gtactctagt ggccgttcca gcgtattcta ctcatagaga tgatgctgtc 1200 tacgctgatg ccttagtatt cgatcctttc agattctcac gtatgagagc gagagaaggt 1260 gaaggtacaa agcaccagtt cgttaatact tcagtcgagt acgttccatt tggtcacgga 1320 aagcatgctt gtccaggaag attcttcgcc gcaaacgaat tgaaagcaat gttggcttac 1380 attgttctaa actatgatgt aaagttgcct ggtgacggta aacgtccatt gaacatgtat 1440 tggggtccaa cagttttgcc tgcaccagca ggccaagtat tgttcagaaa gagacaagtt 1500 agtctataac cgcgg 1515 SEQ ID NO:74 SEQ ID NO:75 atggcatttt tctctatgat ttcaattttg ttgggatttg ttatttcttc tttcatcttc 60 atctttttct tcaaaaagtt acttagtttt agtaggaaaa acatgtcaga agtttctact 120 ttgccaagtg ttccagtagt gcctggtttt ccagttattg ggaatttgtt gcaactaaag 180 gagaaaaagc ctcataaaac tttcactaga tggtcagaga tatatggacc tatctactct 240 ataaagatgg gttcttcatc tcttattgta ttgaacagta cagaaactgc taaggaagca 300 atggtcacta gattttcatc aatatctacc agaaaattgt caaacgccct aacagttcta 360 acctgcgata agtctatggt cgccacttct gattatgatg acttccacaa attagttaag 420 agatgtttgc taaatggact tcttggtgct aatgctcaaa agagaaaaag acactacaga 480 gatgctttga ttgaaaatgt gagttccaag ctacatgcac acgctagaga tcatccacaa 540 gagccagtta actttagagc aattttcgaa cacgaattgt ttggtgtagc attaaagcaa 600 gccttcggta aagacgtaga atccatatac gtcaaggagt taggcgtaac attatcaaaa 660 gatgaaatct ttaaggtgct tgtacatgat atgatggagg gtgcaattga tgtagattgg 720 CA ()2973674 2017-07-12 agagatttct tcccatattt gaaatggatc cctaataagt cttttgaagc taggatacaa 780 caaaagcaca agagaagact agctgttatg aacgcactta tacaggacag attgaagcaa 840 aatgggtctg aatcagatga tgattgttac cttaacttct taatgtctga ggctaaaaca 900 ttgactaagg aacagatcgc aatccttgtc tgggaaacaa tcattgaaac agcagatact 960 accttagtca caactgaatg ggccatatac gagctagcca aacatccatc tgtgcaagat 1020 aggttgtgta aggagatcca gaacgtgtgt ggtggagaga aattcaagga agagcagttg 1080 tcacaagttc cttaccttaa cggcgttttc catgaaacct tgagaaaata ctcacctgca 1140 ccattagttc ctattagata cgcccacgaa gatacacaaa tcggtggcta ccatgttcca 1200 gctgggtccg aaattgctat aaacatctac gggtgcaaca tggacaaaaa gagatgggaa 1260 agaccagaag attggtggcc agaaagattc ttagatgatg gcaaatatga aacatctgat 1320 ttgcataaaa caatggcttt cggagctggc aaaagagtgt gtgccggtgc tctacaagcc 1380 tccctaatgg ctggtatcgc tattggtaga ttggtccaag agttcgaatg gaaacttaga 1440 gatggtgaag aggaaaatgt cgatacttat gggttaacat ctcaaaagtt atacccacta 1500 atggcaatca tcaatcctag aagatcctaa 1530 SEQ ID NO:76 SEQ ID NO:77 atgcaatcag attcagtcaa agtctctcca tttgatttgg tttccgctgc tatgaatggc 60 aaggcaatgg aaaagttgaa cgctagtgaa tctgaagatc caacaacatt gcctgcacta 120 aagatgctag ttgaaaatag agaattgttg acactgttca caacttcctt cgcagttctt 180 attgggtgtc ttgtatttct aatgtggaga cgttcatcct ctaaaaagct ggtacaagat 240 ccagttccac aagttatcgt tgtaaagaag aaagagaagg agtcagaggt tgatgacggg 300 aaaaagaaag tttctatttt ctacggcaca caaacaggaa ctgccgaagg ttttgctaaa 360 gcattagtcg aggaagcaaa agtgagatat gaaaagacct ctttcaaggt tatcgatcta 420 gatgactacg ctgcagatga tgatgaatat gaggaaaaac tgaaaaagga atccttagcc 480 ttcttcttct tggccacata cggtgatggt gaacctactg ataatgctgc taacttctac 540 aagtggttca cagaaggcga cgataaaggt gaatggctga aaaagttaca atacggagta 600 tttggtttag gtaacagaca atatgaacat ttcaacaaga tcgctattgt agttgatgat 660 aaacttactg aaatgggagc caaaagatta gtaccagtag gattagggga tgatgatcag 720 tgtatagaag atgacttcac cgcctggaag gaattggtat ggccagaatt ggatcaactt 780 ttaagggacg aagatgatac ttctgtgact accccataca ctgcagccgt attggagtac 840 agagtggttt accatgataa accagcagac tcatatgctg aagatcaaac ccatacaaac 900 ggtcatgttg ttcatgatgc acagcatcct tcaagatcta atgtggcttt caaaaaggaa 960 ctacacacct ctcaatcaga taggtcttgt actcacttag aattcgatat ttctcacaca 1020 ggactgtctt acgaaactgg cgatcacgtt ggcgtttatt ccgagaactt gtccgaagtt 1080 gtcgatgaag cactaaaact gttagggtta tcaccagaca catacttctc agtccatgct 1140 gataaggagg atgggacacc tatcggtggt gcttcactac caccaccttt tcctccttgc 1200 acattgagag acgctctaac cagatacgca gatgtcttat cctcacctaa aaaggtagct 1260 ttgctggcat tggctgctca tgctagtgat cctagtgaag ccgataggtt aaagttcctg 1320 gcttcaccag ccggaaaaga tgaatatgca caatggatcg tcgccaacca acgttctttg 1380 ctagaagtga tgcaaagttt tccatctgcc aagcctccat taggtgtgtt cttcgcagca 1440 gtagctccac gtttacaacc aagatactac tctatcagtt catctcctaa gatgtctcct 1500 aacagaatac atgttacatg tgctttggtg tacgagacta ctccagcagg cagaattcac 1560 agaggattgt gttcaacctg gatgaaaaat gctgtccctt taacagagtc acctgattgc 1620 tctcaagcat ccattttcgt tagaacatca aatttcagac ttccagtgga tccaaaagtt 1680 ccagtcatta tgataggacc aggcactggt cttgccccat tcaggggctt tcttcaagag 1740 agattggcct tgaaggaatc tggtacagaa ttgggttctt ctatcttttt ctttggttgc 1800 CA ()2973674 2017-07-12 cgtaatagaa aagttgactt tatctacgag gacgagctta acaattttgt tgagacagga 1860 gcattgtcag aattgatcgt cgcattttca agagaaggga ctgccaaaga gtacgttcag 1920 cacaagatga gtcaaaaagc ctccgatata tggaaacttc taagtgaagg tgcctatctt 1980 tatgtctgtg gcgatgcaaa gggcatggcc aaggatgtcc atagaactct gcatacaatt 2040 gttcaggaac aagggagtct ggattcttcc aaggctgaat tgtacgtcaa aaacttacag 2100 atgtctggaa gatacttaag agatgtttgg taa 2133 SEQ ID NO:78 SEQ ID NO:79 atgaaggtca gtccattcga attcatgtcc gctattatca agggtagaat ggacccatct 60 aactcctcat ttgaatctac tggtgaagtt gcctccgtta tctttgaaaa cagagaattg 120 gttgccatct tgaccacttc tattgctgtt atgattggtt gcttcgttgt cttgatgtgg 180 agaagagctg gttctagaaa ggttaagaat gtcgaattgc caaagccatt gattgtccat 240 gaaccagaac ctgaagttga agatggtaag aagaaggttt ccatcttctt cggtactcaa 300 actggtactg ctgaaggttt tgctaaggct ttggctgatg aagctaaagc tagatacgaa 360 aaggctacct tcagagttgt tgatttggat gattatgctg ccgatgatga ccaatacgaa 420 gaaaaattga agaacgaatc cttcgccgtt ttcttgttgg ctacttatgg tgatggtgaa 480 cctactgata atgctgctag attttacaag tggttcgccg aaggtaaaga aagaggtgaa 540 tggttgcaaa acttgcacta tgctgttttt ggtttgggta acagacaata cgaacacttc 600 aacaagattg ctaaggttgc cgacgaatta ttggaagctc aaggtggtaa tagattggtt 660 aaggttggtt taggtgatga cgatcaatgc atcgaagatg atttttctgc ttggagagaa 720 tctttgtggc cagaattgga tatgttgttg agagatgaag atgatgctac tactgttact 780 actccatata ctgctgctgt cttggaatac agagttgtct ttcatgattc tgctgatgtt 840 gctgctgaag ataagtcttg gattaacgct aatggtcatg ctgttcatga tgctcaacat 900 ccattcagat ctaacgttgt cgtcagaaaa gaattgcata cttctgcctc tgatagatcc 960 tgttctcatt tggaattcaa catttccggt tccgctttga attacgaaac tggtgatcat 1020 gttggtgtct actgtgaaaa cttgactgaa actgttgatg aagccttgaa cttgttgggt 1080 ttgtctccag aaacttactt ctctatctac accgataacg aagatggtac tccattgggt 1140 ggttcttcat tgccaccacc atttccatca tgtactttga gaactgcttt gaccagatac 1200 gctgatttgt tgaactctcc aaaaaagtct gctttgttgg ctttagctgc tcatgcttct 1260 aatccagttg aagctgatag attgagatac ttggcttctc cagctggtaa agatgaatat 1320 gcccaatctg ttatcggttc ccaaaagtct ttgttggaag ttatggctga attcccatct 1380 gctaaaccac cattaggtgt tttttttgct gctgttgctc caagattgca acctagattc 1440 tactccattt catcctctcc aagaatggct ccatctagaa tccatgttac ttgtgctttg 1500 gtttacgata agatgccaac tggtagaatt cataagggtg tttgttctac ctggatgaag 1560 aattctgttc caatggaaaa gtcccatgaa tgttcttggg ctccaatttt cgttagacaa 1620 tccaatttta agttgccagc cgaatccaag gttccaatta tcatggttgg tccaggtact 1680 ggtttggctc cttttagagg ttttttacaa gaaagattgg ccttgaaaga atccggtgtt 1740 gaattgggtc catccatttt gtttttcggt tgcagaaaca gaagaatgga ttacatctac 1800 gaagatgaat tgaacaactt cgttgaaacc ggtgctttgt ccgaattggt tattgctttt 1860 tctagagaag gtcctaccaa agaatacgtc caacataaga tggctgaaaa ggcttctgat 1920 atctggaact tgatttctga aggtgcttac ttgtacgttt gtggtgatgc taaaggtatg 1980 gctaaggatg ttcatagaac cttgcatacc atcatgcaag aacaaggttc tttggattct 2040 tccaaagctg aatccatggt caagaacttg caaatgaatg gtagatactt aagagatgtt 2100 CA ()2973674 2017-07-12 tggtaa 2106 SEQ ID NO:80 SEQ ID NO:81 atggcagaat tagatacact tgatatagta gtattaggtg ttatcttttt gggtactgtg 60 gcatacttta ctaagggtaa attgtggggt gttaccaagg atccatacgc taacggattc 120 gctgcaggtg gtgcttccaa gcctggcaga actagaaaca tcgtcgaagc tatggaggaa 180 tcaggtaaaa actgtgttgt tttctacggc agtcaaacag gtacagcgga ggattacgca 240 tcaagacttg caaaggaagg aaagtccaga ttcggtttga acactatgat cgccgatcta 300 gaagattatg acttcgataa cttagacact gttccatctg ataacatcgt tatgtttgta 360 ttggctactt acggtgaagg cgaaccaaca gataacgccg tggatttcta tgagttcatt 420 actggcgaag atgcctcttt caatgagggc aacgatcctc cactaggtaa cttgaattac 480 gttgcgttcg gtctgggcaa caatacctac gaacactaca actcaatggt caggaacgtt 540 aacaaggctc tagaaaagtt aggagctcat agaattggag aagcaggtga gggtgacgac 600 ggagctggaa ctatggaaga ggacttttta gcttggaaag atccaatgtg ggaagccttg 660 gctaaaaaga tgggcttgga ggaaagagaa gctgtatatg aacctatttt cgctatcaat 720 gagagagatg atttgacccc tgaagcgaat gaggtatact tgggagaacc taataagcta 780 cacttggaag gtacagcgaa aggtccattc aactcccaca acccatatat cgcaccaatt 840 gcagaatcat acgaactttt ctcagctaag gatagaaatt gtctgcatat ggaaattgat 900 atttctggta gtaatctaaa gtatgaaaca ggcgaccata tcgcgatctg gcctaccaac 960 ccaggtgaag aggtcaacaa atttcttgac attctagatc tgtctggtaa gcaacattcc 1020 gtcgtaacag tgaaagcctt agaacctaca gccaaagttc cttttccaaa tccaactacc 1080 tacgatgcta tattgagata ccatctggaa atatgcgctc cagtttctag acagtttgtc 1140 tcaactttag cagcattcgc ccctaatgat gatatcaaag ctgagatgaa ccgtttggga 1200 tcagacaaag attacttcca cgaaaagaca ggaccacatt actacaatat cgctagattt 1260 ttggcctcag tctctaaagg tgaaaaatgg acaaagatac cattttctgc tttcatagaa 1320 ggccttacaa aactacaacc aagatactat tctatctctt cctctagttt agttcagcct 1380 aaaaagatta gtattactgc tgttgtcgaa tctcagcaaa ttccaggtag agatgaccca 1440 ttcagaggtg tagcgactaa ctacttgttc gctttgaagc agaaacaaaa cggtgatcca 1500 aatccagctc cttttggcca atcatacgag ttgacaggac caaggaataa gtatgatggt 1560 atacatgttc cagtccatgt aagacattct aactttaagc taccatctga tccaggcaaa 1620 cctattatca tgatcggtcc aggtaccggt gttgcccctt ttagaggctt cgtccaagag 1680 agggcaaaac aagccagaga tggtgtagaa gttggtaaaa cactgctgtt ctttggatgt 1740 agaaagagta cagaagattt catgtatcaa aaagagtggc aagagtacaa ggaagctctt 1800 ggcgacaaat tcgaaatgat tacagctttt tcaagagaag gatctaaaaa ggtttatgtt 1860 caacacagac tgaaggaaag atcaaaggaa gtttctgatc ttctatccca aaaagcatac 1920 ttctacgttt gcggagacgc cgcacatatg gcacgtgaag tgaacactgt gttagcacag 1980 atcatagcag aaggccgtgg tgtatcagaa gccaagggtg aggaaattgt caaaaacatg 2040 agatcagcaa atcaatacca agtgtgttct gatttcgtaa ctttacactg taaagagaca 2100 acatacgcga attcagaatt gcaagaggat gtctggagtt aa 2142 SEQ ID NO:82 CA ()2973674 2017-07-12 SEQ ID NO:83 atgcaatcgg aatccgttga agcatcgacg attgatttga tgactgctgt tttgaaggac 60 acagtgatcg atacagcgaa cgcatctgat aacggagact caaagatgcc gccggcgttg 120 gcgatgatgt tcgaaattcg tgatctgttg ctgattttga ctacgtcagt tgctgttttg 180 gtcggatgtt tcgttgtttt ggtgtggaag agatcgtccg ggaagaagtc cggcaaggaa 240 ttggagccgc cgaagatcgt tgtgccgaag aggcggctgg agcaggaggt tgatgatggt 300 aagaagaagg ttacgatttt cttcggaaca caaactggaa cggctgaagg tttcgctaag 360 gcacttttcg aagaagcgaa agcgcgatat gaaaaggcag cgtttaaagt gattgatttg 420 gatgattatg ctgctgattt ggatgagtat gcagagaagc tgaagaagga aacatatgct 480 ttcttcttct tggctacata tggagatggt gagccaactg ataatgctgc caaattttat 540 aaatggttta ctgagggaga cgagaaaggc gtttggcttc aaaaacttca atatggagta 600 tttggtcttg gcaacagaca atatgaacat ttcaacaaga ttggaatagt ggttgatgat 660 ggtctcaccg agcagggtgc aaaacgcatt gttcccgttg gtcttggaga cgacgatcaa 720 tcaattgaag acgatttttc ggcatggaaa gagttagtgt ggcccgaatt ggatctattg 780 cttcgcgatg aagatgacaa agctgctgca actccttaca cagctgcaat ccctgaatac 840 cgcgtcgtat ttcatgacaa acccgatgcg ttttctgatg atcatactca aaccaatggt 900 catgctgttc atgatgctca acatccatgc agatccaatg tggctgttaa aaaagagctt 960 catactcctg aatccgatcg ttcatgcaca catcttgaat ttgacatttc tcacactgga 1020 ttatcttatg aaactgggga tcatgttggt gtatactgtg aaaacctaat tgaagtagtg 1080 gaagaagctg ggaaattgtt aggattatca acagatactt atttctcgtt acatattgat 1140 aacgaagatg gttcaccact tggtggacct tcattacaac ctccttttcc tccttgtact 1200 ttaagaaaag cattgactaa ttatgcagat ctgttaagct ctcccaaaaa gtcaactttg 1260 cttgctctag ctgctcatgc ttccgatccc actgaagctg atcgtttaag atttcttgca 1320 tctcgcgagg gcaaggatga atatgctgaa tgggttgttg caaaccaaag aagtcttctt 1380 gaagtcatgg aagctttccc gtcagctaga ccgccacttg gtgttttctt tgcagcggtt 1440 gcaccgcgtt tacagcctcg ttactactct atttcttcct ccccaaagat ggaaccaaac 1500 aggattcatg ttacttgcgc gttggtttat gaaaaaactc ccgcaggtcg tatccacaaa 1560 ggaatctgct caacctggat gaagaacgct gtacctttga ccgaaagtca agattgcagt 1620 tgggcaccga tttttgttag aacatcaaac ttcagacttc caattgaccc gaaagtcccg 1680 gttatcatga ttggtcctgg aaccgggttg gctccattta ggggttttct tcaagaaaga 1740 ttggctctta aagaatccgg aaccgaactc gggtcatcta ttttattctt cggttgtaga 1800 aaccgcaaag tggattacat atatgagaat gaactcaaca actttgttga aaatggtgcg 1860 ctttctgagc ttgatgttgc tttctcccgc gatggcccga cgaaagaata cgtgcaacat 1920 aaaatgaccc aaaaggcttc tgaaatatgg aatatgcttt ctgagggagc atatttatat 1980 gtatgtggtg atgctaaagg catggctaaa gatgtacacc gtacacttca caccattgtg 2040 caagaacagg gaagtttgga ctcgtctaaa gcggagttgt atgtgaagaa tctacaaatg 2100 tcaggaagat acctccgtga tgtttggtaa 2130 SEQ ID NO:84 CA ()2973674 2017-07-12 SEQ ID NO:85 atgcaatcta actccgtgaa gatttcgccg cttgatctgg taactgcgct gtttagcggc 60 aaggttttgg acacatcgaa cgcatcggaa tcgggagaat ctgctatgct gccgactata 120 gcgatgatta tggagaatcg tgagctgttg atgatactca caacgtcggt tgctgtattg 180 atcggatgcg ttgtcgtttt ggtgtggcgg agatcgtcta cgaagaagtc ggcgttggag 240 ccaccggtga ttgtggttcc gaagagagtg caagaggagg aagttgatga tggtaagaag 300 aaagttacgg ttttcttcgg cacccaaact ggaacagctg aaggcttcgc taaggcactt 360 gttgaggaag ctaaagctcg atatgaaaag gctgtcttta aagtaattga tttggatgat 420 tatgctgctg atgacgatga gtatgaggag aaactaaaga aagaatcttt ggcctttttc 480 tttttggcta cgtatggaga tggtgagcca acagataatg ctgccagatt ttataaatgg 540 tttactgagg gagatgcgaa aggagaatgg cttaataagc ttcaatatgg agtatttggt 600 ttgggtaaca gacaatatga acattttaac aagatcgcaa aagtggttga tgatggtctt 660 gtagaacagg gtgcaaagcg tcttgttcct gttggacttg gagatgatga tcaatgtatt 720 gaagatgact tcaccgcatg gaaagagtta gtatggccgg agttggatca attacttcgt 780 gatgaggatg acacaactgt tgctactcca tacacagctg ctgttgcaga atatcgcgtt 840 gtttttcatg aaaaaccaga cgcgctttct gaagattata gttatacaaa tggccatgct 900 gttcatgatg ctcaacatcc atgcagatcc aacgtggctg tcaaaaagga acttcatagt 960 cctgaatctg accggtcttg cactcatctt gaatttgaca tctcgaacac cggactatca 1020 tatgaaactg gggaccatgt tggagtttac tgtgaaaact tgagtgaagt tgtgaatgat 1080 gctgaaagat tagtaggatt accaccagac acttactcct ccatccacac tgatagtgaa 1140 gacgggtcgc cacttggcgg agcctcattg ccgcctcctt tcccgccatg cactttaagg 1200 aaagcattga cgtgttatgc tgatgttttg agttctccca agaagtcggc tttgcttgca 1260 ctagctgctc atgccaccga tcccagtgaa gctgatagat tgaaatttct tgcatccccc 1320 gccggaaagg atgaatattc tcaatggata gttgcaagcc aaagaagtct ccttgaagtc 1380 atggaagcat tcccgtcagc taagccttca cttggtgttt tctttgcatc tgttgccccg 1440 cgcttacaac caagatacta ctctatttct tcctcaccca agatggcacc ggataggatt 1500 catgttacat gtgcattagt ctatgagaaa acacctgcag gccgcatcca caaaggagtt 1560 tgttcaactt ggatgaagaa cgcagtgcct atgaccgaga gtcaagattg cagttgggcc 1620 ccaatatacg tccgaacatc caatttcaga ctaccatctg accctaaggt cccggttatc 1680 atgattggac ctggcactgg tttggctcct tttagaggtt tccttcaaga gcggttagct 1740 ttaaaggaag ccggaactga cctcggttta tccattttat tcttcggatg taggaatcgc 1800 aaagtggatt tcatatatga aaacgagctt aacaactttg tggagactgg tgctctttct 1860 gagcttattg ttgctttctc ccgtgaaggc ccgactaagg aatatgtgca acacaagatg 1920 agtgagaagg cttcggatat ctggaacttg ctttctgaag gagcatattt atacgtatgt 1980 ggtgatgcca aaggcatggc caaagatgta catcgaaccc tccacacaat tgtgcaagaa 2040 cagggatctc ttgactcgtc aaaggcagaa ctctacgtga agaatctaca aatgtcagga 2100 agatacctcc gtgacgtttg gtaa 2124 SEQ ID NO:86 CA ()2973674 2017-07-12 SEQ ID NO:87 atgtcctcca actccgattt ggtcagaaga ttggaatctg ttttgggtgt ttctttcggt 60 ggttctgtta ctgattccgt tgttgttatt gctaccacct ctattgcttt ggttatcggt 120 gttttggttt tgttgtggag aagatcctct gacagatcta gagaagttaa gcaattggct 180 gttccaaagc cagttactat cgttgaagaa gaagatgaat tcgaagttgc ttctggtaag 240 accagagttt ctattttcta cggtactcaa actggtactg ctgaaggttt tgctaaggct 300 ttggctgaag aaatcaaagc cagatacgaa aaagctgccg ttaaggttat tgatttggat 360 gattacacag ccgaagatga caaatacggt gaaaagttga agaaagaaac tatggccttc 420 ttcatgttgg ctacttatgg tgatggtgaa cctactgata atgctgctag attttacaag 480 tggttcaccg aaggtactga tagaggtgtt tggttggaac atttgagata cggtgtattc 540 ggtttgggta acagacaata cgaacacttc aacaagattg ccaaggttgt tgatgatttg 600 ttggttgaac aaggtgccaa gagattggtt actgttggtt tgggtgatga tgatcaatgc 660 atcgaagatg atttctccgc ttggaaagaa gccttgtggc cagaattgga tcaattattg 720 caagatgata ccaacaccgt ttctactcca tacactgctg ttattccaga atacagagtt 780 gttatccacg atccatctgt tacctcttat gaagatccat actctaacat ggctaacggt 840 aatgcctctt acgatattca tcatccatgt agagctaacg ttgccgtcca aaaagaattg 900 cataagccag aatctgacag aagttgcatc catttggaat tcgatatttt cgctactggt 960 ttgacttacg aaaccggtga tcatgttggt gtttacgctg ataattgtga tgatactgta 1020 gaagaagccg ctaagttgtt gggtcaacca ttggatttgt tgttctccat tcataccgat 1080 aacaacgacg gtacttcttt gggttcttct ttgccaccac catttccagg tccatgtact 1140 ttgagaactg ctttggctag atatgccgat ttgttgaatc caccaaaaaa ggctgctttg 1200 attgctttag ctgctcatgc tgatgaacca tctgaagctg aaagattgaa gttcttgtca 1260 tctccacaag gtaaggacga atattctaaa tgggttgtcg gttcccaaag atccttggtt 1320 gaagttatgg ctgaatttcc atctgctaaa ccaccattgg gtgtattttt tgctgctgtt 1380 gttcctagat tgcaacctag atattactcc atctcttcca gtccaagatt tgctccacat 1440 agagttcatg ttacttgcgc tttggtttat ggtccaactc caactggtag aattcacaga 1500 ggtgtatgtt cattctggat gaagaatgtt gtcccattgg aaaagtctca aaactgttct 1560 tgggccccaa ttttcatcag acaatctaat ttcaagttgc cagccgatca ttctgttcca 1620 atagttatgg ttggtccagg tactggttta gctcctttta gaggtttctt acaagaaaga 1680 ttggccttga aagaagaagg tgctcaagtt ggtcctgctt tgttgttttt tggttgcaga 1740 aacagacaaa tggacttcat ctacgaagtc gaattgaaca actttgtcga acaaggtgct 1800 ttgtccgaat tgatcgttgc tttttcaaga gaaggtccat ccaaagaata cgtccaacat 1860 aagatggttg aaaaggcagc ttacatgtgg aacttgattt ctcaaggtgg ttacttctac 1920 gtttgtggtg atgctaaagg tatggctaga gatgttcata gaacattgca taccatcgtc 1980 caacaagaag aaaaggttga ttctaccaag gccgaatcca tcgttaagaa attgcaaatg 2040 gacggtagat acttgagaga tgtttggtga 2070 SEQ ID NO:88 SEQ ID NO:89 atgacttctg cactttatgc ctccgatctt ttcaaacaat tgaaaagtat catgggaacg 60 gattctttgt ccgatgatgt tgtattagtt attgctacaa cttctctggc actggttgct 120 ggtttcgttg tcttattgtg gaaaaagacc acggcagatc gttccggcga gctaaagcca 180 ctaatgatcc ctaagtctct gatggcgaaa gatgaggatg atgacttaga tctaggttct 240 CA ()2973674 2017-07-12 ggaaaaacga gagtctctat cttcttcggc acacaaaccg gaacagccga aggattcgct 300 aaagcacttt cagaagagat caaagcaaga tacgaaaagg cggctgtaaa agtaatcgat 360 ttggatgatt acgctgccga tgatgaccaa tatgaggaaa agttgaaaaa ggaaacattg 420 gctttctttt gtgtagccac gtatggtgat ggtgaaccaa ccgataacgc cgcaagattc 480 tacaagtggt ttactgaaga gaacgaaaga gatatcaagt tgcagcaact tgcttacggc 540 gtttttgcct taggtaacag acaatacgag cactttaaca agataggtat tgtcttagat 600 gaagagttat gcaaaaaggg tgcgaagaga ttgattgaag tcggtttagg agatgatgat 660 caatctatcg aggatgactt taatgcatgg aaggaatctt tgtggtctga attagataag 720 ttacttaagg acgaagatga taaatccgtt gccactccat acacagccgt cattccagaa 780 tatagagtag ttactcatga tccaagattc acaacacaga aatcaatgga aagtaatgtg 840 gctaatggta atactaccat cgatattcat catccatgta gagtagacgt tgcagttcaa 900 aaggaattgc acactcatga atcagacaga tcttgcatac atcttgaatt tgatatatca 960 cgtactggta tcacttacga aacaggtgat cacgtgggtg tctacgctga aaaccatgtt 1020 gaaattgtag aggaagctgg aaagttgttg ggccatagtt tagatcttgt tttctcaatt 1080 catgccgata aagaggatgg ctcaccacta gaaagtgcag tgcctccacc atttccagga 1140 ccatgcaccc taggtaccgg tttagctcgt tacgcggatc tgttaaatcc tccacgtaaa 1200 tcagctctag tggccttggc tgcgtacgcc acagaacctt ctgaggcaga aaaactgaaa 1260 catctaactt caccagatgg taaggatgaa tactcacaat ggatagtagc tagtcaacgt 1320 tctttactag aagttatggc tgctttccca tccgctaaac ctcctttggg tgttttcttc 1380 gccgcaatag cgcctagact gcaaccaaga tactattcaa tttcatcctc acctagactg 1440 gcaccatcaa gagttcatgt cacatccgct ttagtgtacg gtccaactcc tactggtaga 1500 atccataagg gcgtttgttc aacatggatg aaaaacgcgg ttccagcaga gaagtctcac 1560 gaatgttctg gtgctccaat ctttatcaga gcctccaact tcaaactgcc ttccaatcct 1620 tctactccta ttgtcatggt cggtcctggt acaggtcttg ctccattcag aggtttctta 1680 caagagagaa tggccttaaa ggaggatggt gaagagttgg gatcttcttt gttgtttttc 1740 ggctgtagaa acagacaaat ggatttcatc tacgaagatg aactgaataa ctttgtagat 1800 caaggagtta tttcagagtt gataatggct ttttctagag aaggtgctca gaaggagtac 1860 gtccaacaca aaatgatgga aaaggccgca caagtttggg acttaatcaa agaggaaggc 1920 tatctatatg tctgtggtga tgcaaagggt atggcaagag atgttcacag aacacttcat 1980 actatagtcc aggaacagga aggcgttagt tcttctgaag cggaagcaat tgtgaaaaag 2040 ttacaaacag agggaagata cttgagagat gtgtggtaa 2079 SEQ ID NO:90 SEQ ID NO:91 atgtcttcct cttcctcttc cagtacctct atgattgatt tgatggctgc tattattaaa 60 ggtgaaccag ttatcgtctc cgacccagca aatgcctctg cttatgaatc agttgctgca 120 gaattgtctt caatgttgat cgaaaacaga caattcgcca tgatcgtaac tacatcaatc 180 gctgttttga tcggttgtat tgtcatgttg gtatggagaa gatccggtag tggtaattct 240 aaaagagtcg aacctttgaa accattagta attaagccaa gagaagaaga aatagatgac 300 ggtagaaaga aagttacaat atttttcggt acccaaactg gtacagctga aggttttgca 360 aaagccttag gtgaagaagc taaggcaaga tacgaaaaga ctagattcaa gatagtcgat 420 ttggatgact atgccgctga tgacgatgaa tacgaagaaa agttgaagaa agaagatgtt 480 gcatttttct ttttggcaac ctatggtgac ggtgaaccaa ctgacaatgc agccagattc 540 tacaaatggt ttacagaggg taatgatcgt ggtgaatggt tgaaaaactt aaagtacggt 600 CA ()2973674 2017-07-12 gttttcggtt tgggtaacag acaatacgaa catttcaaca aagttgcaaa ggttgtcgac 660 gatattttgg tcgaacaagg tgctcaaaga ttagtccaag taggtttggg tgacgatgac 720 caatgtatag aagatgactt tactgcctgg agagaagctt tgtggcctga attagacaca 780 atcttgagag aagaaggtga caccgccgtt gctaccccat atactgctgc agtattagaa 840 tacagagttt ccatccatga tagtgaagac gcaaagttta atgatatcac tttggccaat 900 ggtaacggtt atacagtttt cgatgcacaa cacccttaca aagctaacgt tgcagtcaag 960 agagaattac atacaccaga atccgacaga agttgtatac acttggaatt tgatatcgct 1020 ggttccggtt taaccatgaa gttgggtgac catgtaggtg ttttatgcga caatttgtct 1080 gaaactgttg atgaagcatt gagattgttg gatatgtccc ctgacactta ttttagtttg 1140 cacgctgaaa aagaagatgg tacaccaatt tccagttctt taccacctcc attccctcca 1200 tgtaacttaa gaacagcctt gaccagatac gcttgcttgt tatcatcccc taaaaagtcc 1260 gccttggttg ctttagccgc tcatgctagt gatcctactg aagcagaaag attgaaacac 1320 ttagcatctc cagccggtaa agatgaatat tcaaagtggg tagttgaatc tcaaagatca 1380 ttgttagaag ttatggcaga atttccatct gccaagcctc cattaggtgt cttctttgct 1440 ggtgtagcac ctagattgca accaagattc tactcaatca gttcttcacc taagatcgct 1500 gaaactagaa ttcatgttac atgtgcatta gtctacgaaa agatgccaac cggtagaatt 1560 cacaagggtg tatgctctac ttggatgaaa aatgctgttc cttacgaaaa atcagaaaag 1620 ttgttcttag gtagaccaat cttcgtaaga caatcaaact tcaagttgcc ttctgattca 1680 aaggttccaa taatcatgat aggtcctggt acaggtttag ccccattcag aggtttcttg 1740 caagaaagat tggctttagt tgaatctggt gtcgaattag gtccttcagt tttgttcttt 1800 ggttgtagaa acagaagaat ggatttcatc tatgaagaag aattgcaaag attcgtcgaa 1860 tctggtgcat tggccgaatt atctgtagct ttttcaagag aaggtccaac taaggaatac 1920 gttcaacata agatgatgga taaggcatcc gacatatgga acatgatcag tcaaggtgct 1980 tatttgtacg tttgcggtga cgcaaagggt atggccagag atgtccatag atctttgcac 2040 acaattgctc aagaacaagg ttccatggat agtaccaaag ctgaaggttt cgtaaagaac 2100 ttacaaactt ccggtagata cttgagagat gtctggtga 2139 SEQ ID NO:92 SEQ ID NO:93 atggaagcct cttacctata catttctatt ttgcttttac tggcatcata cctgttcacc 60 actcaactta gaaggaagag cgctaatcta ccaccaaccg tgtttccatc aataccaatc 120 attggacact tatacttact caaaaagcct ctttatagaa ctttagcaaa aattgccgct 180 aagtacggac caatactgca attacaactc ggctacagac gtgttctggt gatttcctca 240 ccatcagcag cagaagagtg ctttaccaat aacgatgtaa tcttcgcaaa tagacctaag 300 acattgtttg gcaaaatagt gggtggaaca tcccttggca gtttatccta cggcgatcaa 360 tggcgtaatc taaggagagt agcttctatc gaaatcctat cagttcatag gttgaacgaa 420 tttcatgata tcagagtgga tgagaacaga ttgttaatta gaaaacttag aagttcatct 480 tctcctgtta ctcttataac agtcttttat gctctaacat tgaacgtcat tatgagaatg 540 atctctggca aaagatattt cgacagtggg gatagagaat tggaggagga aggtaagaga 600 tttcgagaaa tcttagacga aacgttgctt ctagccggtg cttctaatgt tggcgactac 660 ttaccaatat tgaactggtt gggagttaag tctcttgaaa agaaattgat cgctttgcag 720 aaaaagagag atgacttttt ccagggtttg attgaacagg ttagaaaatc tcgtggtgct 780 aaagtaggca aaggtagaaa aacgatgatc gaactcttat tatctttgca agagtcagaa 840 cctgagtact atacagatgc tatgataaga tcttttgtcc taggtctgct ggctgcaggt 900 CA ()2973674 2017-07-12 agtgatactt cagcgggcac tatggaatgg gccatgagct tactggtcaa tcacccacat 960 gtattgaaga aagctcaagc tgaaatcgat agagttatcg gtaataacag attgattgac 1020 gagtcagaca ttggaaatat cccttacatc gggtgtatta tcaatgaaac tctaagactc 1080 tatccagcag ggccattgtt gttcccacat gaaagttctg ccgactgcgt tatttccggt 1140 tacaatatac ctagaggtac aatgttaatc gtaaaccaat gggcgattca tcacgatcct 1200 aaagtctggg atgatcctga aacctttaaa cctgaaagat ttcaaggatt agaaggaact 1260 agagatggtt tcaaacttat gccattcggt tctgggagaa gaggatgtcc aggtgaaggt 1320 ttggcaataa ggctgttagg gatgacacta ggctcagtga tccaatgttt tgattgggag 1380 agagtaggag atgagatggt tgacatgaca gaaggtttgg gtgtcacact tcctaaggcc 1440 gttccattag ttgccaaatg taagccacgt tccgaaatga ctaatctcct atccgaactt 1500 taa 1503 SEQ ID NO:94 SEQ ID NO:95 atggaagtaa cagtagctag tagtgtagcc ctgagcctgg tctttattag catagtagta 60 agatgggcat ggagtgtggt gaattgggtg tggtttaagc cgaagaagct ggaaagattt 120 ttgagggagc aaggccttaa aggcaattcc tacaggtttt tatatggaga catgaaggag 180 aactctatcc tgctcaaaca agcaagatcc aaacccatga acctctccac ctcccatgac 240 atagcacctc aagtcacccc ttttgtcgac caaaccgtga aagcttacgg taagaactct 300 tttaattggg ttggccccat accaagggtg aacataatga atccagaaga tttgaaggac 360 gtcttaacaa aaaatgttga ctttgttaag ccaatatcaa acccacttat caagttgcta 420 gctacaggta ttgcaatcta tgaaggtgag aaatggacta aacacagaag gattatcaac 480 ccaacattcc attcggagag gctaaagcgt atgttacctt catttcacca aagttgtaat 540 gagatggtca aggaatggga gagcttggtg tcaaaagagg gttcatcatg tgagttggat 600 gtctggcctt ttcttgaaaa tatgtcggca gatgtgatct cgagaacagc atttggaact 660 agctacaaaa aaggacagaa aatctttgaa ctcttgagag agcaagtaat atatgtaacg 720 aaaggctttc aaagttttta cattccagga tggaggtttc tcccaactaa gatgaacaag 780 aggatgaatg agattaacga agaaataaaa ggattaatca ggggtattat aattgacaga 840 gagcaaatca ttaaggcagg tgaagaaacc aacgatgact tattaggtgc acttatggag 900 tcaaacttga aggacattcg ggaacatggg aaaaacaaca aaaatgttgg gatgagtatt 960 gaagatgtaa ttcaggagtg taagctgttt tactttgctg ggcaagaaac cacttcagtg 1020 ttgctggctt ggacaatggt tttacttggt caaaatcaga actggcaaga tcgagcaaga 1080 caagaggttt tgcaagtctt tggaagcagc aagccagatt ttgatggtct agctcacctt 1140 aaagtcgtaa ccatgatttt gcttgaagtt cttcgattat acccaccagt cattgaactt 1200 attcgaacca ttcacaagaa aacacaactt gggaagctct cactaccaga aggagttgaa 1260 gtccgcttac caacactgct cattcaccat gacaaggaac tgtggggtga tgatgcaaac 1320 cagttcaatc cagagaggtt ttcggaagga gtttccaaag caacaaagaa ccgactctca 1380 ttcttcccct tcggagccgg tccacgcatt tgcattggac agaacttttc tatgatggaa 1440 gcaaagttgg ccttagcatt gatcttgcaa cacttcacct ttgagctttc tccatctcat 1500 gcacatgctc cttcccatcg tataaccctt caaccacagt atggtgttcg tatcatttta 1560 catcgacgtt ag 1572 SEQ ID NO:96 atggaagtca ctgtcgcctc ttctgtcgct ttatccttag tcttcatttc cattgtcgtc 60 agatgggctt ggtccgttgt caactgggtt tggttcaaac caaagaagtt ggaaagattc 120 ttgagagagc aaggtttgaa gggtaattct tatagattct tgtacggtga catgaaggaa 180 aattctattt tgttgaagca agccagatcc aaaccaatga acttgtctac ctctcatgat 240 CA ()2973674 2017-07-12 attgctccac aagttactcc attcgtcgat caaactgtta aagcctacgg taagaactct 300 ttcaattggg ttggtccaat tcctagagtt aacatcatga acccagaaga tttgaaggat 360 gtcttgacca agaacgttga cttcgttaag ccaatttcca acccattgat taaattgttg 420 gctactggta ttgccattta cgaaggtgaa aagtggacta agcatagaag aatcatcaac 480 cctaccttcc actctgaaag attgaagaga atgttaccat ctttccatca atcctgtaat 540 gaaatggtta aggaatggga atccttggtt tctaaagaag gttcttcttg cgaattggat 600 gtttggccat tcttggaaaa tatgtctgct gatgtcattt ccagaaccgc tttcggtacc 660 tcctacaaga agggtcaaaa gattttcgaa ttgttgagag agcaagttat ttacgttacc 720 aagggtttcc aatccttcta catcccaggt tggagattct tgccaactaa aatgaacaag 780 cgtatgaacg agatcaacga agaaattaaa ggtttgatca gaggtattat tatcgacaga 840 gaacaaatta ttaaagctgg tgaagaaacc aacgatgatt tgttgggtgc tttgatggag 900 tccaacttga aggatattag agaacatggt aagaacaaca agaatgttgg tatgtctatt 960 gaagatgtta ttcaagaatg taagttattc tacttcgctg gtcaagagac cacttctgtt 1020 ttgttagcct ggactatggt cttgttaggt caaaaccaaa attggcaaga tagagctaga 1080 caagaagttt tgcaagtctt cggttcttcc aagccagact ttgatggttt ggcccacttg 1140 aaggttgtta ctatgatttt gttagaagtt ttgagattgt acccaccagt cattgagtta 1200 atcagaacca ttcataaaaa gactcaattg ggtaaattat ctttgccaga aggtgttgaa 1260 gtcagattac caaccttgtt gattcaccac gataaggaat tatggggtga cgacgctaat 1320 caatttaatc cagaaagatt ttccgaaggt gtttccaagg ctaccaaaaa ccgtttgtcc 1380 ttcttcccat ttggtgctgg tccacgtatt tgtatcggtc aaaacttttc catgatggaa 1440 gccaagttgg ctttggcttt aatcttgcaa cacttcactt tcgaattgtc tccatcccat 1500 gcccacgctc cttctcatag aatcacttta caaccacaat acggtgtcag aatcatctta 1560 cacagaagat aa 1572 SEQ ID NO:97 SEQ ID NO:98 atggaagcat caagggctag ttgtgttgcg ctatgtgttg tttgggtgag catagtaatt 60 acattggcat ggagggtgct gaattgggtg tggttgaggc caaagaaact agaaagatgc 120 ttgagggagc aaggccttac aggcaattct tacaggcttt tgtttggaga caccaaggat 180 ctctcgaaga tgctggaaca aacacaatcc aaacccatca aactctccac ctcccatgat 240 atagcgccac gagtcacccc atttttccat cgaactgtga actctaatgg caagaattct 300 tttgtttgga tgggccctat accaagagtg cacatcatga atccagaaga tttgaaagat 360 gccttcaaca gacatgatga ttttcataag acagtaaaaa atcctatcat gaagtctcca 420 ccaccgggca ttgtaggcat tgaaggtgag caatgggcta aacacagaaa gattatcaac 480 ccagcattcc atttagagaa gctaaagggt atggtaccaa tattttacca aagttgtagc 540 gagatgatta acaaatggga gagcttggtg tccaaagaga gttcatgtga gttggatgtg 600 tggccttatc ttgaaaattt taccagcgat gtgatttccc gagctgcatt tggaagtagc 660 tatgaagagg gaaggaaaat atttcaacta ctaagagagg aagcaaaagt ttattcggta 720 gctctacgaa gtgtttacat tccaggatgg aggtttctac caaccaagca gaacaagaag 780 acgaaggaaa ttcacaatga aattaaaggc ttacttaagg gcattataaa taaaagggaa 840 gaggcgatga aggcagggga agccactaaa gatgacttac taggaatact tatggagtcc 900 aacttcaggg aaattcagga acatgggaac aacaaaaatg ctggaatgag tattgaagat 960 gtaattggag agtgtaagtt gttttacttt gctgggcaag agaccacttc ggtgttgctt 1020 gtttggacaa tgattttact aagccaaaat caggattggc aagctcgtgc aagagaagag 1080 gtcttgaaag tctttggaag caacatccca acctatgaag agctaagtca cctaaaagtt 1140 gtgaccatga ttttacttga agttcttcga ttatacccat cagtcgttgc gcttcctcga 1200 accactcaca agaaaacaca gcttggaaaa ttatcattac cagctggagt ggaagtctcc 1260 CA ()2973674 2017-07-12 ttgcccatac tgcttgttca ccatgacaaa gagttgtggg gtgaggatgc aaatgagttc 1320 aagccagaga ggttttcaga gggagtttca aaggcaacaa agaacaaatt tacatactta 1380 cctttcggag ggggtccaag gatttgcatt ggacaaaact ttgccatggt ggaagctaaa 1440 ttggccttgg ccctgatttt acaacacttt gcctttgagc tttctccatc ctatgctcat 1500 gctccttctg cagttataac ccttcaacct caatttggtg ctcatatcat tttgcataaa 1560 cgttga 1566 SEQ ID NO:99 atggaagctt ctagagcatc ttgtgttgct ttgtgtgttg tttgggtttc catcgttatt 60 actttggctt ggagagtttt gaattgggtc tggttaagac caaaaaagtt ggaaagatgc 120 ttgagagaac aaggtttgac tggtaactct tacagattgt tgttcggtga taccaaggac 180 ttgtctaaga tgttggaaca aactcaatcc aagcctatca agttgtctac ctctcatgat 240 attgctccaa gagttactcc attcttccat agaactgtta actccaacgg taagaactct 300 tttgtttgga tgggtccaat tccaagagtc catattatga accctgaaga tttgaaggac 360 gctttcaaca gacatgatga tttccataag accgtcaaga acccaattat gaagtctcca 420 ccaccaggta tagttggtat tgaaggtgaa caatgggcca aacatagaaa gattattaac 480 ccagccttcc acttggaaaa gttgaaaggt atggttccaa tcttctacca atcctgctct 540 gaaatgatta acaagtggga atccttggtt tccaaagaat cttcctgtga attggatgtc 600 tggccatatt tggaaaactt cacctccgat gttatttcca gagctgcttt tggttcttct 660 tacgaagaag gtagaaagat cttccaatta ttgagagaag aagccaaggt ttactccgtt 720 gctttgagat ctgtttacat tccaggttgg agattcttgc caactaagca aaacaaaaag 780 accaaagaaa tccacaacga aatcaagggt ttgttgaagg gtatcatcaa caagagagaa 840 gaagctatga aggctggtga agctacaaaa gatgatttgt tgggtatctt gatggaatcc 900 aacttcagag aaatccaaga acacggtaac aacaagaatg ccggtatgtc tattgaagat 960 gttatcggtg aatgcaagtt gttctacttt gctggtcaag aaactacctc cgttttgttg 1020 gtttggacca tgattttgtt gtcccaaaat caagattggc aagctagagc tagagaagaa 1080 gtcttgaaag ttttcggttc taacatccca acctacgaag aattgtctca cttgaaggtt 1140 gtcactatga tcttgttgga agtattgaga ttatacccat ccgttgttgc attgccaaga 1200 actactcata agaaaactca attgggtaaa ttgtccttgc cagctggtgt tgaagtttct 1260 ttgccaattt tgttagtcca ccacgacaaa gaattgtggg gtgaagatgc taatgaattc 1320 aagccagaaa gattctccga aggtgtttct aaagctacca agaacaagtt cacttacttg 1380 ccatttggtg gtggtccaag aatatgtatt ggtcaaaatt tcgctatggt cgaagctaaa 1440 ttggctttgg ctttgatctt gcaacatttc gctttcgaat tgtcaccatc ttatgctcat 1500 gctccatctg ctgttattac attgcaacca caatttggtg cccatatcat cttgcataag 1560 agataac 1567 SEQ ID NO:100 SEQ ID NO:101 CA ()2973674 2017-07-12 SEQ ID NO:102 SEQ ID NO:103 SEQ ID NO:104 SEQ ID NO:105 atgggtttgt tcccattaga ggattcctac gcgctggtct ttgaaggact agcaataaca 60 ctggctttgt actatctact gtctttcatc tacaaaacat ctaaaaagac atgtacacct 120 cctaaagcat ctggtgaaat cattccaatt acaggaatca tattgaatct gctatctggc 180 tcaagtggtc tacctattat cttagcactt gcctctttag cagacagatg tggtcctatt 240 ttcaccatta ggctgggtat taggagagtg ctagtagtat caaattggga aatcgctaag 300 gagattttca ctacccacga tttgatagtt tctaatagac caaaatactt agccgctaag 360 attcttggtt tcaattatgt ttcattctct ttcgctccat acggcccata ttgggtcgga 420 atcagaaaga ttattgctac aaaactaatg tcttcttcca gacttcagaa gttgcaattt 480 gtaagagttt ttgaactaga aaactctatg aaatctatca gagaatcatg gaaggagaaa 540 aaggatgaag agggaaaggt attagttgag atgaaaaagt ggttctggga actgaatatg 600 aacatagtgt taaggacagt tgctggtaaa caatacactg gtacagttga tgatgccgat 660 gcaaagcgta tctccgagtt attcagagaa tggtttcact acactggcag atttgtcgtt 720 ggagacgctt ttccttttct aggttggttg gacctgggcg gatacaaaaa gacaatggaa 780 ttagttgcta gtagattgga ctcaatggtc agtaaatggt tagatgagca tcgtaaaaag 840 caagctaacg atgacaaaaa ggaggatatg gatttcatgg atatcatgat ctccatgaca 900 gaagcaaatt caccacttga aggatacggc actgatacta ttatcaagac cacatgtatg 960 actttgattg tttcaggagt tgatacaacc tcaatcgtac ttacttgggc cttatcactt 1020 ttgttaaaca acagagatac tttgaaaaag gcacaagagg aattagatat gtgcgtaggt 1080 aaaggaagac aagtcaacga gtctgatctt gttaacttga tatacttgga agcagtgctt 1140 aaagaggctt taagacttta cccagcagcg ttcttaggcg gaccaagagc attcttggaa 1200 gattgtactg ttgctggtta tagaattcca aagggcacct gcttgttgat taacatgtgg 1260 aaactgcata gagatccaaa catttggagt gatccttgcg aattcaagcc agaaagattt 1320 ttgacaccta atcaaaagga tgttgatgtg atcggtatgg atttcgaatt gataccattt 1380 ggtgccggca gaagatattg tccaggtact agattggctt tacagatgtt gcatatcgta 1440 CA ()2973674 2017-07-12 ttagcgacat tgctgcaaaa cttcgaaatg tcaacaccaa acgatgcgcc agtcgatatg 1500 actgcttctg ttggcatgac aaatgccaaa gcatcacctt tagaagtctt gctatcacct 1560 cgtgttaaat ggtcctaa 1578 SEQ ID NO:106 SEQ ID NO:107 atgatacaag ttttaactcc aattctactc ttcctcatct tcttcgtttt ctggaaagtc 60 tacaaacatc aaaagactaa aatcaatcta ccaccaggtt ccttcggctg gccatttttg 120 ggtgaaacct tagccttact tagagcaggc tgggattctg agccagaaag attcgtaaga 180 gagcgtatca aaaagcatgg atctccactt gttttcaaga catcactatt tggagacaga 240 ttcgctgttc tttgcggtcc agctggtaat aagtttttgt tctgcaacga aaacaaatta 300 gtggcatctt ggtggccagt ccctgtaagg aagttgttcg gtaaaagttt actcacaata 360 agaggagatg aagcaaaatg gatgagaaaa atgctattgt cttacttggg tccagatgca 420 tttgccacac attatgccgt tactatggat gttgtaacac gtagacatat tgatgtccat 480 tggaggggca aggaggaagt taatgtattt caaacagtta agttgtacgc attcgaatta 540 gcttgtagat tattcatgaa cctagatgac ccaaaccaca tcgcgaaact cggtagtctt 600 ttcaacattt tcctcaaagg gatcatcgag cttcctatag acgttcctgg aactagattt 660 tactccagta aaaaggccgc agctgccatt agaattgaat tgaaaaagct cattaaagct 720 agaaaactcg aattgaagga gggtaaggcg tcttcttcac aggacttgct ttctcatcta 780 ttaacatcac ctgatgagaa tgggatgttc ttgacagaag aggaaatagt cgataacatt 840 ctacttttgt tattcgctgg tcacgatacc tctgcactat caataacact tttgatgaaa 900 accttaggtg aacacagtga tgtgtacgac aaggttttga aggaacaatt agaaatttcc 960 aaaacaaagg aggcttggga atcactaaag tgggaagata tccagaagat gaagtactca 1020 tggtcagtaa tctgtgaagt catgagattg aatcctcctg tcatagggac atacagagag 1080 gcgttggttg atatcgacta tgctggttac actatcccaa aaggatggaa gttgcattgg 1140 tcagctgttt ctactcaaag agacgaagcc aatttcgaag atgtaactag attcgatcca 1200 tccagatttg aaggggcagg ccctactcca ttcacatttg tgcctttcgg tggaggtcct 1260 agaatgtgtt taggcaaaga gtttgccagg ttagaagtgt tagcatttct ccacaacatt 1320 gttaccaact ttaagtggga tcttctaatc cctgatgaga agatcgaata tgatccaatg 1380 gctactccag ctaagggctt gccaattaga cttcatccac accaagtcta a 1431 SEQ ID NO:108 SEQ ID NO:109 atggagtctt tagtggttca tacagtaaat gctatctggt gtattgtaat cgtcgggatt 60 ttctcagttg gttatcacgt ttacggtaga gctgtggtcg aacaatggag aatgagaaga 120 tcactgaagc tacaaggtgt taaaggccca ccaccatcca tcttcaatgg taacgtctca 180 gaaatgcaac gtatccaatc cgaagctaaa cactgctctg gcgataacat tatctcacat 240 gattattctt cttcattatt cccacacttc gatcactgga gaaaacagta cggcagaatc 300 CA ()2973674 2017-07-12 tacacatact ctactggatt aaagcaacac ttgtacatca atcatccaga aatggtgaag 360 gagctatctc agactaacac attgaacttg ggtagaatca cccatataac caaaagattg 420 aatcctatct taggtaacgg aatcataacc tctaatggtc ctcattgggc ccatcagcgt 480 agaattatcg cctacgagtt tactcatgat aagatcaagg gtatggttgg tttgatggtt 540 gagtctgcta tgcctatgtt gaataagtgg gaggagatgg taaagagagg cggagaaatg 600 ggatgcgaca taagagttga tgaggacttg aaagatgttt cagcagatgt gattgcaaaa 660 gcctgtttcg gatcctcatt ttctaaaggt aaggctattt tctctatgat aagagatttg 720 cttacagcta tcacaaagag aagtgttcta ttcagattca acggattcac tgatatggtc 780 tttgggagta aaaagcatgg tgacgttgat atagacgctt tagaaatgga attggaatca 840 tccatttggg aaactgtcaa ggaacgtgaa atagaatgta aagatactca caaaaaggat 900 ctgatgcaat tgattttgga aggggcaatg cgttcatgtg acggtaacct ttgggataaa 960 tcagcatata gaagatttgt tgtagataat tgtaaatcta tctacttcgc agggcatgat 1020 agtacagctg tctcagtgtc atggtgtttg atgttactgg ccctaaaccc atcatggcaa 1080 gttaagatcc gtgatgaaat tctgtcttct tgcaaaaatg gtattccaga tgccgaaagt 1140 atcccaaacc ttaaaacagt gactatggtt attcaagaga caatgagatt ataccctcca 1200 gcaccaatcg tcgggagaga agcctctaaa gatatcagat tgggcgatct agttgttcct 1260 aaaggcgtct gtatatggac actaatacca gctttacaca gagatcctga gatttgggga 1320 ccagatgcaa acgatttcaa accagaaaga ttttctgaag gaatttcaaa ggcttgtaag 1380 tatcctcaaa gttacattcc atttggtctg ggtcctagaa catgcgttgg taaaaacttt 1440 ggcatgatgg aagtaaaggt tcttgtttcc ctgattgtct ccaagttctc tttcactcta 1500 tctcctacct accaacatag tcctagtcac aaacttttag tagaaccaca acatggggtg 1560 gtaattagag tggtttaa 1578 SEQ ID NO:110 SEQ ID NO:111 atgtacttcc tactacaata cctcaacatc acaaccgttg gtgtctttgc cacattgttt 60 ctctcttatt gtttacttct ctggagaagt agagcgggta acaaaaagat tgccccagaa 120 gctgccgctg catggcctat tatcggccac ctccacttac ttgcaggtgg atcccatcaa 180 ctaccacata ttacattggg taacatggca gataagtacg gtcctgtatt cacaatcaga 240 ataggcttgc atagagctgt agttgtctca tcttgggaaa tggcaaagga atgttcaaca 300 gctaatgatc aagtgtcttc ttcaagacct gaactattag cttctaagtt gttgggttat 360 aactacgcca tgtttggttt ttcaccatac ggttcatact ggagagaaat gagaaagatc 420 atctctctcg aattactatc taattccaga ttggaactat tgaaagatgt tagagcctca 480 gaagttgtca catctattaa ggaactatac aaattgtggg cggaaaagaa gaatgagtca 540 ggattggttt ctgtcgagat gaaacaatgg ttcggagatt tgactttaaa cgtgatcttg 600 agaatggtgg ctggtaaaag atacttctcc gcgagtgacg cttcagaaaa caaacaggcc 660 cagcgttgta gaagagtctt cagagaattc ttccatctct ccggcttgtt tgtggttgct 720 gatgctatac cttttcttgg atggctcgat tggggaagac acgagaagac cttgaaaaag 780 accgccatag aaatggattc catcgcccag gagtggcttg aggaacatag acgtagaaaa 840 gattctggag atgataattc tacccaagat ttcatggacg ttatgcaatc tgtgctagat 900 ggcaaaaatc taggcggata cgatgctgat acgattaaca aggctacatg cttaactctt 960 atatcaggtg gcagtgatac tactgtagtt tctttgacat gggctcttag tcttgtgtta 1020 aacaatagag atactttgaa aaaggcacag gaagagttag acatccaagt cggtaaggaa 1080 agattggtta acgagcaaga catcagtaag ttagtttact tgcaagcaat agtaaaagag 1140 acactcagac tttatccacc aggtcctttg ggtggtttga gacaattcac tgaagattgt 1200 acactaggtg gctatcacgt ttcaaaagga actagattaa tcatgaactt atccaagatt 1260 caaaaagatc cacgtatttg gtctgatcct actgaattcc aaccagagag attccttacg 1320 CA ()2973674 2017-07-12 actcataaag atgtcgatcc acgtggtaaa cactttgaat tcattccatt cggtgcagga 1380 agacgtgcat gtcctggtat cacattcgga ttacaagtac tacatctaac attggcatct 1440 ttcttgcatg cgtttgaatt ttcaacacca tcaaatgagc aggttaacat gagagaatca 1500 ttaggtctta cgaatatgaa atctacccca ttagaagttt tgatttctcc aagactatcc 1560 cttaattgct tcaaccttat gaaaatttga 1590 SEQ ID NO:112 SEQ ID NO:113 atggaaccta acttttactt gtcattacta ttgttgttcg tgaccttcat ttctttaagt 60 ctgtttttca tcttttacaa acaaaagtcc ccattgaatt tgccaccagg gaaaatgggt 120 taccctatca taggtgaaag tttagaattc ctatccacag gctggaaggg acatcctgaa 180 aagttcatat ttgatagaat gcgtaagtac agtagtgagt tattcaagac ttctattgta 240 ggcgaatcca cagttgtttg ctgtggggca gctagtaaca aattcctatt ctctaacgaa 300 aacaaactgg taactgcctg gtggccagat tctgttaaca aaatcttccc aacaacttca 360 ctggattcta atttgaagga ggaatctata aagatgagaa agttgctgcc acagttcttc 420 aaaccagaag cacttcaaag atacgtcggc gttatggatg taatcgcaca aagacatttt 480 gtcactcact gggacaacaa aaatgagatc acagtttatc cacttgctaa aagatacact 540 ttcttgcttg cgtgtagact gttcatgtct gttgaggatg aaaatcatgt ggcgaaattc 600 tcagacccat tccaactaat cgctgcaggc atcatttcac ttcctatcga tcttcctggt 660 actccattca acaaggccat aaaggcttca aatttcatta gaaaagagct gataaagatt 720 atcaaacaaa gacgtgttga tctggcagag ggtacagcat ctccaaccca ggatatcttg 780 tcacatatgc tattaacatc tgatgaaaac ggtaaatcta tgaacgagtt gaacattgcc 840 gacaagattc ttggactatt gataggaggc cacgatacag cttcagtagc ttgcacattt 900 ctagtgaagt acttaggaga attaccacat atctacgata aagtctacca agagcaaatg 960 gaaattgcca agtccaaacc tgctggggaa ttgttgaatt gggatgactt gaaaaagatg 1020 aagtattcat ggaatgtggc atgtgaggta atgagattgt caccaccttt acaaggtggt 1080 tttagagagg ctataactga ctttatgttt aacggtttct ctattccaaa agggtggaag 1140 ttatactggt ccgccaactc tacacacaaa aatgcagaat gtttcccaat gcctgagaaa 1200 ttcgatccta ccagatttga aggtaatggt ccagcgcctt atacatttgt accattcggt 1260 ggaggcccta gaatgtgtcc tggaaaggaa tacgctagat tagaaatctt ggttttcatg 1320 cataatctgg tcaaacgttt taagtgggaa aaggttattc cagacgaaaa gattattgtc 1380 gatccattcc caatcccagc taaagatctt ccaatccgtt tgtatcctca caaagcttaa 1440 SEQ ID NO:114 SEQ ID NO:115 atggcctctg ttactttggg ttcctggatc gtcgtccacc accataacca tcaccatcca 60 tcatctatcc taactaaatc tcgttcaaga tcctgtccta ttacactaac caaaccaatc 120 tcttttcgtt caaagagaac agtttcctct agtagttcta tcgtgtcctc tagtgtcgtc 180 CA ()2973674 2017-07-12 actaaggaag acaatctgag acagtctgaa ccttcttcct ttgatttcat gtcatatatc 240 attactaagg cagaactagt gaataaggct cttgattcag cagttccatt aagagagcca 300 ttgaaaatcc atgaagcaat gagatactct cttctagctg gcgggaagag agtcagacct 360 gtactctgca tagcagcgtg cgaattagtt ggtggcgagg aatcaaccgc tatgcctgcc 420 gcttgtgctg tagaaatgat tcatacaatg tcactgatac acgatgattt gccatgtatg 480 gataacgatg atctgagaag gggtaagcca actaaccata aggttttcgg cgaagatgtt 540 gccgtcttag ctggtgatgc tttgttatct ttcgcgttcg aacatttggc atccgcaaca 600 tcaagtgatg ttgtgtcacc agtaagagta gttagagcag ttggagaact ggctaaagct 660 attggaactg agggtttagt tgcaggtcaa gtcgtcgata tctcttccga aggtcttgat 720 ttgaatgatg taggtcttga acatctcgaa ttcatccatc ttcacaagac agctgcactt 780 ttagaagcca gtgcggttct cggcgcaatt gttggcggag ggagtgatga cgaaattgag 840 agattgagga agtttgctag atgtatagga ttactgttcc aagtagtaga cgatatacta 900 gatgtgacaa agtcttccaa agagttggga aaaacagctg gtaaagattt gattgccgac 960 aaattgacct accctaagat tatggggcta gaaaaatcaa gagaatttgc cgagaaactc 1020 aatagagagg cgcgtgatca actgttgggt ttcgattctg ataaagttgc accactctta 1080 gccttagcca actacatcgc ttacagacaa aactaa 1116 SEQ ID NO:116 SEQ ID NO:117 SEQ ID NO:118 SEQ ID NO:119 CA ()2973674 2017-07-12 SEQ ID NO:120 SEQ ID NO:121 SEQ ID NO:122 SEQ ID NO:123 SEQ ID NO:124 SEQ ID NO:125 CA ()2973674 2017-07-12 SEQ ID NO:126 SEQ ID NO:127 SEQ ID NO:128 SEQ ID NO:129 SEQ ID NO:130 SEQ ID NO:131 SEQ ID NO:132 SEQ ID NO:133 SEQ ID NO:134 SEQ ID NO:135 ggcaagccac gtttggtg 18 SEQ ID NO:136 ggagctgcat gtgtcagagg 20 SEQ ID NO:137 cgatgtattt catcactggt tgccatccat cgcggct 37 SEQ ID NO:138 agccgcgatg gatggcaacc agtgatgaaa tacatcg 37 SEQ ID NO:139 ttatgattat actcactact gggctgctgc agccgcattg 40 SEQ ID NO:140 agccgcgatg gatggcaacc agtgatgaaa tacatcg 37 SEQ ID NO:141 caaacctatt actttccttg gtttactgcc accggaaata c 41 SEQ ID NO:142 gtatttccgg tggcagtaaa ccaaggaaag taataggttt g 41 SEQ ID NO:143 ccggtggttc cggtgggact aatgcctcca ttacatga 38 SEQ ID NO:144 tcatgtaatg gaggcattag tcccaccgga accaccgg 38 SEQ ID NO:145 gaacgcaggt ctgcaggttc caagaaatga ggaagatgg 39 SEQ ID NO:146 ccatcttcct catttcttgg aacctgcaga cctgcgttc 39 SEQ ID NO:147 SEQ ID NO:148 SEQ ID NO:149 SEQ ID NO:150 CA ()2973674 2017-07-12 SEQ ID NO:151 SEQ ID NO:152 SEQ ID NO:153 SEQ ID NO:154 SEQ ID NO:155 CA ()2973674 2017-07-12 SEQ ID NO:156 SEQ ID NO:157 SEQ ID NO:158 SEQ ID NO:159 SEQ ID NO:160 CA ()2973674 2017-07-12 SEQ ID NO:161 SEQ ID NO:162 SEQ ID NO:163 SEQ ID NO:164 SEQ ID NO:165 SEQ ID NO:166 CA ()2973674 2017-07-12 SEQ ID NO:167 SEQ ID NO:168 SEQ ID NO:169 SEQ ID NO:170 SEQ ID NO:171 CA ()2973674 2017-07-12 SEQ ID NO:172 SEQ ID NO:173 SEQ ID NO:174 SEQ ID NO:175 SEQ ID NO:176 CA ()2973674 2017-07-12 SEQ ID NO:177 SEQ ID NO:178 SEQ ID NO:179 SEQ ID NO:180 SEQ ID NO:181 CA ()2973674 2017-07-12 SEQ ID NO:182 SEQ ID NO:183 SEQ ID NO:184 SEQ ID NO:185 SEQ ID NO:186 SEQ ID NO:187 CA ()2973674 2017-07-12 SEQ ID NO:188 SEQ ID NO:189 SEQ ID NO:190 SEQ ID NO:191 SEQ ID NO:192 CA ()2973674 2017-07-12 SEQ ID NO:193 SEQ ID NO:194 SEQ ID NO:195 SEQ ID NO:196 SEQ ID NO:197 SEQ ID NO:198 CA ()2973674 2017-07-12 SEQ ID NO:199 SEQ ID NO:200 SEQ ID NO:201 SEQ ID NO:202 SEQ ID NO:203 SEQ ID NO:204 CA ()2973674 2017-07-12 SEQ ID NO:205 SEQ ID NO:206 SEQ ID NO:207 SEQ ID NO:208 SEQ ID NO:209 SEQ ID NO:210 CA ()2973674 2017-07-12 SEQ ID NO:211 SEQ ID NO:212 SEQ ID NO:213 SEQ ID NO:214 SEQ ID NO:215 ATCAACGGGUAAAATGGATGCTATGGCTACCACCG
SEQ ID NO:216 CGTGCGAUTCAGTTTCTGGCCAAAACGGTGATT
SEQ ID NO:217 CA ()2973674 2017-07-12 SEQ ID NO:218 SEQ ID NO:219 SEQ ID NO:220

Claims (42)

WHAT IS CLAIMED IS:
1. A recombinant host cell, comprising at least one recombinant gene that is:
(a) a gene encoding a UGT91D2e polypeptide having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:11;
(b) a gene encoding a chimeric polypeptide having at least 70% sequence identity to the amino acid sequence set forth in SEQ ID NO:17 or SEQ ID
NO:18;
(c) a gene encoding a UGT85C2 polypeptide having at least 55% sequence identity to the amino acid sequence set forth in SEQ ID NO:7; and/or (d) a gene encoding a UGT76G1 polypeptide having at least 50% sequence identity to the amino acid sequence set forth in SEQ ID NO:9;
wherein the recombinant host cell is capable of producing a steviol glycoside, glycosylated ent-kaurenol compound, and/or a glycosylated ent-kaurenoic acid compound in a cell culture broth.
2. The recombinant host cell of claim 1, wherein the UGT91D2e polypeptide comprises a UGT91D2e polypeptide having at least one amino acid substitution at residues 93, 99, 114, 144, 148, 152, 195, 196, 199, 211, 213, 221, 286, 384, 426, 438, or 466 of SEQ ID
NO:11.
3. The recombinant host cell of claim 1, wherein the UGT85C2 polypeptide comprises a UGT85C2 polypeptide having at least one amino acid substitution at residues 21, 48, 49, 84, 86, 87, 91, 92, 95, 122, 334, or 334 of SEQ ID NO:7.
4. The recombinant host cell of claim 1, wherein the UGT76G1 polypeptide comprises a UGT76G1 polypeptide having at least one amino acid substitution at residues 23, 26, 55, 146, 257, 283, and 337 of SEQ ID NO:9.
5. The recombinant host cell of claim 1, wherein the UGT91D2e polypeptide comprises one or more of the UGT91D2e polypeptide variants comprising: P93V, S99I, S114F, T144K, T144L, T144M, A148K, M152T, L195G, L195C, L1955, L195N, L195V, V196P, K199C, L211H, L211M, L211I, L211C, L211T, L213E, S221I, V286C, V286N, V2865, G384W, G384K, G384Y, E426G, E438H, 3438M or A466V of SEQ ID NO:11.
6. The recombinant host cell of claim 1, wherein the UGT85C2 polypeptide comprises one or more of the UGT85C2 polypeptide variants comprising: Q21L, Q21T, Q21V, F48S, F48H, F48Y, F48R, F48Q, F48W, F48T, I49V, S84G, S84A, S84T, S84C, S84P, S84N, S84V, P86R, P86G, I87H, I87P, I87M, I87Y, L91K, L91R, L91T, L92F, L92I, L92M, I95K, F122S, L334S or L334M of SEQ ID NO:7.
7. The recombinant host cell of claim 1, wherein the UGT76G1 polypeptide comprises one or more of the UGT76G1 polypeptide variants comprising: Q23H, I26W, T146G, H155L, L257G, S253W, T284G, 5283N, K337P or T55K of SEQ ID NO:9.
8. The recombinant host cell of any one of claims 1-7, further comprising at least one recombinant gene that is:
(a) a gene encoding a geranylgeranyl diphosphate synthase (GGPPS) polypeptide;
(b) a gene encoding an ent-copalyl diphosphate synthase (CDPS) polypeptide;
(c) a gene encoding an ent-kaurene synthase (KS) polypeptide;
(d) a gene encoding an ent-kaurene oxidase (KO) polypeptide;
(e) a gene encoding a cytochrome P450 reductase (CPR) polypeptide; and (f) a gene encoding an ent-kaurenoic acid hydroxylase (KAH) polypeptide;
(g) a gene encoding a UGT74G1 polypeptide; and/or (h) a gene encoding an EUGT11 polypeptide;
wherein the recombinant host cell capable of producing a steviol glycoside, glycosylated ent-kaurenol compound, and/or a glycosylated ent-kaurenoic acid compound in a cell culture broth.
9. The recombinant host cell of claim 8, wherein:
(a) the GGPPS polypeptide comprises a polypeptide having at least 70%
identity to an amino acid sequence set forth in SEQ ID NO:20, SEQ ID
NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, or SEQ ID NO:116;

(b) the CDPS polypeptide comprises a polypeptide having at least 70%
identity to an amino acid sequence set forth in SEQ ID NO:34, SEQ ID
NO:36, SEQ ID NO:38, SEQ ID NO:40, or SEQ ID NO:42;
(c) the KS polypeptide comprises a polypeptide having at least 70% identity to an amino acid sequence set forth in SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50, or SEQ ID NO:52;
(d) the KO polypeptide comprises a polypeptide having at least 70% identity to an amino acid sequence set forth in SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:117, SEQ ID NO:66, SEQ ID NO:68, SEQ ID NO:70, SEQ ID
NO:72, SEQ ID NO:74, or SEQ ID NO:76;
(e) the CPR polypeptide comprises a polypeptide having at least 70%
identity to an amino acid sequence set forth in SEQ ID NO:78, SEQ ID NO:80, SEQ ID NO:82, SEQ ID NO:84, SEQ ID NO:86, SEQ ID NO:88, SEQ ID
NO:90, SEQ ID NO:92;
(f) the KAH polypeptide comprises a polypeptide having at least 70%
identity to an amino acid sequence set forth in SEQ ID NO:94, SEQ ID NO:97, SEQ ID NO:100, SEQ ID NO:101, SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO:104, SEQ ID NO:106, SEQ ID NO:108, SEQ ID NO:110, SEQ ID NO:112, or SEQ ID NO:114;
(g) the UGT74G1 polypeptide comprises a polypeptide having at least 55%
identity to an amino acid sequence set forth in SEQ ID NO:4;
(h) the EUGT11 polypeptide comprises a polypeptide having at least 65%
identity to an amino acid sequence set forth in SEQ ID NO:16.
10. The recombinant host cell of any one of claims 1-9, wherein the cell culture broth comprises:
(a) the steviol glycoside, glycosylated ent-kaurenol compound, and/or the glycosylated ent-kaurenoic acid compound produced by the recombinant host cell, (b) glucose, fructose and/or sucrose; and/or (c) supplemental nutrients comprising trace metals, vitamins, salts, yeast nitrogen base (YNB), and/or amino acids.
11. The recombinant host cell of any one of claims 1-10, wherein the recombinant host comprises a plant cell, a mammalian cell, an insect cell, a fungal cell, an algal cell, or a bacterial cell.
12. The recombinant host cell of claim 11, wherein the bacterial cell comprises Escherichia cells, Lactobacillus cells, Lactococcus cells, Cornebacterium cells, Acetobacter cells, Acinetobacter cells, or Pseudomonas cells.
13. The recombinant host cell of claim 11, wherein the fungal cell comprises a yeast cell.
14. The recombinant host cell of claim 13, wherein the yeast cell is a cell from Saccharomyces cerevisiae, Schizosaccharomyces pombe, Yarrowia lipolytica, Candida glabrata, Ashbya gossypii, Cyberlindnera jadinii, Pichia pastoris, Kluyveromyces lactis, Hansenula polymorpha, Candida boidinii, Arxula adeninivorans, Xanthophyllomyces dendrorhous, or Candida albicans species.
15. The recombinant host cell of claim 14, wherein the yeast cell is a Saccharomycete.
16. The recombinant host cell of claim 15, wherein the yeast cell is a cell from the Saccharomyces cerevisiae species.
17. A method of producing a steviol glycoside, glycosylated ent-kaurenol compound, and/or glycosylated ent-kaurenoic acid compound in a cell culture broth, comprising growing the recombinant host cell of any one of claims 1-16 in a culture medium, under conditions in which one or more of the genes are expressed;
wherein at least one of the genes is a recombinant gene;
wherein the steviol glycoside, glycosylated ent-kaurenol compound, and/or the glycosylated ent-kaurenoic acid compound is produced by the recombinant host cell.
18. The method of claim 17, wherein one or more of the genes is constitutively expressed and/or expression of one or more of the genes is induced.
19. A method for producing a steviol glycoside, glycosylated ent-kaurenol compound, and/or the glycosylated ent-kaurenoic acid compound comprising whole-cell bioconversion of plant-derived components or synthetic steviol or steviol glycosides using one or more of:
(a) a UGT91D2e polypeptide having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:11;
(b) a chimeric polypeptide having at least 70% sequence identity to the amino acid sequence set forth in SEQ ID NO:17 or SEQ ID NO:18;
(c) a UGT85C2 polypeptide having at least 55% sequence identity to the amino acid sequence set forth in SEQ ID NO:7; and/or (d) a UGT76G1 polypeptide having at least 50% sequence identity to the amino acid sequence set forth in SEQ ID NO:9;
wherein at least one of the polypeptides is a recombinant polypeptide.
20. The method of claim 19, wherein the whole cell is the recombinant host cell of any one of claims 1-16.
21. The method of any one of claims 17-20, wherein the recombinant host cell is grown in a fermentor at a temperature for a period of time, wherein the temperature and period of time facilitate the production of the steviol glycoside, glycosylated ent-kaurenol compound, and/or glycosylated ent-kaurenoic acid compound.
22. An in vitro method for producing a steviol glycoside, glycosylated ent-kaurenol compound, and/or the glycosylated ent-kaurenoic acid compound, comprising adding one or more of:
(a) a UGT91D2e polypeptide having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:11;
(b) a chimeric polypeptide having at least 70% sequence identity to the amino acid sequence set forth in SEQ ID NO:17 or SEQ ID NO:18;
(c) a UGT85C2 polypeptide having at least 55% sequence identity to the amino acid sequence set forth in SEQ ID NO:7; and/or (d) a UGT76G1 polypeptide having at least 50% sequence identity to the amino acid sequence set forth in SEQ ID NO:9, and plant-derived components or synthetic steviol or steviol glycosides to a reaction mixture;

wherein at least one of the polypeptides is a recombinant polypeptide; and synthesizing the steviol glycoside, glycosylated ent-kaurenol compound, and/or the glycosylated ent-kaurenoic acid compound in the reaction mixture.
23. The method of any one of claims 17-22, that further comprises isolating the steviol glycoside, glycosylated ent-kaurenol compound, and/or the glycosylated ent-kaurenoic acid compound, alone or in combination from the cell culture broth.
24. The method of claim 23, wherein the isolating step comprises:
(a) providing the cell culture broth comprising the steviol glycoside, glycosylated ent-kaurenol compound, and/or the glycosylated ent-kaurenoic acid compound alone or in combination;
(b) separating a liquid phase of the cell culture broth from a solid phase of the cell culture broth to obtain a supernatant comprising the steviol glycoside, glycosylated ent-kaurenol compound, and/or the glycosylated ent-kaurenoic acid compound alone or in combination;
(c) providing one or more adsorbent resins, comprising providing the adsorbent resins in a packed column; and (d) contacting the supernatant of step (b) with the one or more adsorbent resins in order to obtain at least a portion of the steviol glycoside, glycosylated ent-kaurenol compound, and/or the glycosylated ent-kaurenoic acid compound alone or in combination thereby isolating the steviol glycoside, glycosylated ent-kaurenol compound, and/or the glycosylated ent-kaurenoic acid compound alone or in combination.
25. The method of any one of claims 17-22, that further comprises recovering the the steviol glycoside, glycosylated ent-kaurenol compound, and/or the glycosylated ent-kaurenoic acid compound alone or a composition comprising the steviol glycoside, glycosylated ent-kaurenol compound, and/or the glycosylated ent-kaurenoic acid compound.
26. The method of claim 25, wherein the recovered composition is enriched for the steviol glycoside, glycosylated ent-kaurenol compound, and/or the glycosylated ent-kaurenoic acid compound relative to a steviol glycoside composition of Stevie plant and has a reduced level of non-steviol glycoside Stevia plant-derived components relative to a plant-derived stevia extract.
27. The method of any one of claims 17-22, wherein the cell culture broth comprises:
(a) one or more steviol glycosides, glycosylated ent-kaurenol compounds, and/or glycosylated ent-kaurenoic acid compounds produced by the recombinant host cell of any one of claims 1-12, (b) glucose, fructose, and/or sucrose; and/or (c) supplemental nutrients comprising trace metals, vitamins, salts, YNB, and/or amino acids.
28. The method of claim 22, wherein the reaction mixture comprising:
(a) one or more steviol glycosides, glycosylated ent-kaurenol compounds, and/or a glycosylated ent-kaurenoic acid compounds produced in the reaction mixture;
(b) a UGT polypeptide;
(c) UDP-glucose, UDP-rhamnose, UDP-xylose, and/or N-acetyl-glucosamine; and/or (d) reaction buffer and/or salts.
29. The method of any one of claims 17-28, wherein the recombinant host cell comprises a plant cell, a mammalian cell, an insect cell, a fungal cell, an algal cell, or a bacterial cell.
30. The method of claim 29, wherein the bacterial cell comprises Escherichia cells, Lactobacillus cells, Lactococcus cells, Cornebacterium cells, Acetobacter cells, Acinetobacter cells, or Pseudomonas cells.
31. The method of claim 29, wherein the fungal cell comprises a yeast cell.
32. The method of claim 31, wherein the yeast cell is a cell from Saccharomyces cerevisiae, Schizosaccharomyces pombe, Yarrowia lipolytica, Candida glabrata, Ashbya gossypii, Cyberlindnera jadinii, Pichia pastoris, Kluyveromyces lactis, Hansenula polymorpha, Candida boidinii, Arxula adeninivorans, Xanthophyllomyces dendrorhous, or Candida albicans species.
33. The method of claim 31, wherein the yeast cell is a Saccharomycete.
34. The method of claim 33, wherein the yeast cell is a cell from the Saccharomyces cerevisiae species.
35. The recombinant host cell of any one of claims 1-16 or the method of any one of claims 17-34, wherein:
(a) the steviol glycoside comprises 13-SMG, 19-SMG, Stevio1-1,2-bioside, Stevio1-1,3-bioside, 1,2-stevioside, 1,3-stevioside, rubusoside, RebA, RebB, RebD, RebE, RebM, di-glycosylated tri-glycosylated steviol, tetra-glycosylated steviol, penta-glycosylated steviol, hexa-glycosylated steviol, hepta-glycosylated steviol, and/or isomers thereof;
(b) the glycosylated ent-kaurenol compound comprises di-glycosylated ent-kaurenol, tri-glycosylated ent-kaurenol, and/or isomers thereof; and/or (c) the glycosylated ent-kaurenoic acid compound comprises di-glycosylated ent-kaurenoic acid, tri-glycosylated ent-kaurenoic acid, and/or isomers thereof.
36. The recombinant host cell or the method of claim 35, wherein:
(a) the di-glycosylated steviol comprises compound 2.23 of Table 1;
(b) the tri-glycosylated steviol comprises compound 3.1 and/or compound 3.34 of Table 1;
(c) the tetra-glycosylated steviol comprises compound 4.26 and/or compound 4.33 of Table 1;
(d) the penta-glycosylated steviol comprises compound 5.22, compound 5.24, and/or compound 5.25 of Table 1;
(e) the hexa-glycosylated steviol comprises compound 6.1 and/or compound 6.23 of Table 1;
(f) the hepta-glycosylated steviol comprises compound 7.2, compound 7.5, and/or compound 7.13 of Table 1;
(9) the glycosylated ent-kaurenoic acid compound comprises compound KA3.1, compound KA3.2, and/or compound KA2.7 of Table 1; and/or (h) the glycosylated ent-kaurenol compound comprises compound KL2.8 and/or compound KL3.1 co-eluted with compound KL3.6 of Table 1.
37. The recombinant host cell or method of claim 36, wherein:
(a) compound 4.26 has the structure:
(b) compound 5.22 has the structure:
(c) compound 6.1 has the structure:

(d) compound 7.2 has the structure:
(e) compound 7.5 has the structure:
(f) compound KA3.1 has the structure:

(g) compound KA3.2 has the structure:
(h) compound KL3.1 has the structure:
38. A steviol glycoside composition produced by the recombinant host cell of any one of claims 1-16 or the method of any one of claims 17-34, wherein the composition has a steviol glycoside composition enriched for RebD, RebM, or isomers thereof relative to a steviol glycoside composition of Stevie plant and has a reduced level of non-steviol glycoside Stevie plant-derived components relative to a plant-derived stevia extract.
39. A cell culture broth comprising:
(a) the recombinant host cell of any one of claims 1-16; and (b) one or more steviol glycosides, glycosylated ent-kaurenol compounds, and/or glycosylated ent-kaurenoic acid compounds produced by the recombinant host cell;
wherein one or more steviol glycosides is present at a concentration of at least 1 mg/liter of the culture broth.
40. A cell culture broth comprising:
(a) one or more steviol glycosides, glycosylated ent-kaurenol compounds, and/or glycosylated ent-kaurenoic acid compounds produced by the recombinant host cell of any one of claims 1-16, (b) glucose, fructose, sucrose, xylose, ethanol, and/or glycerol; and/or (c) supplemental nutrients comprising trace metals, vitamins, salts, YNB, and/or amino acids.
41. A cell lysate comprising:
(a) one or more steviol glycosides, glycosylated ent-kaurenol compounds, and/or glycosylated ent-kaurenoic acid compounds produced by the recombinant host cell of any one of claims 1-16, (b) glucose, fructose, sucrose, xylose, ethanol, glycerol, uridine diphosphate (UDP)-glucose, UDP-rhamnose, UDP-xylose, and/or N-acetyl-glucosamine; and/or (c) supplemental nutrients comprising trace metals, vitamins, salts, YNB, and/or amino acids.
42. A reaction mixture comprising:
(a) one or more steviol glycosides, glycosylated ent-kaurenol compounds, and/or a glycosylated ent-kaurenoic acid compounds produced in the reaction mixture;
(b) a UGT polypeptide;
(c) glucose, fructose, sucrose, xylose, ethanol, glycerol, uridine diphosphate (UDP)-glucose, UDP-rhamnose, UDP-xylose, and/or N-acetyl-glucosamine; and/or (d) reaction buffer and/or salts.
CA2973674A 2015-01-30 2016-01-29 Production of steviol glycosides in recombinant hosts Pending CA2973674A1 (en)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
US201562110207P 2015-01-30 2015-01-30
US62/110,207 2015-01-30
PCT/EP2016/052007 WO2016120486A1 (en) 2015-01-30 2016-01-29 Production of steviol glycosides in recombinant hosts

Publications (1)

Publication Number Publication Date
CA2973674A1 true CA2973674A1 (en) 2016-08-04

Family

ID=55398256

Family Applications (1)

Application Number Title Priority Date Filing Date
CA2973674A Pending CA2973674A1 (en) 2015-01-30 2016-01-29 Production of steviol glycosides in recombinant hosts

Country Status (9)

Country Link
US (4) US10364450B2 (en)
EP (2) EP4148137A1 (en)
JP (1) JP2018503388A (en)
CN (1) CN108337892B (en)
AU (2) AU2016211124B2 (en)
BR (1) BR112017016338A2 (en)
CA (1) CA2973674A1 (en)
SG (1) SG11201705606PA (en)
WO (1) WO2016120486A1 (en)

Families Citing this family (36)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
NZ708078A (en) 2010-06-02 2017-01-27 Evolva Nutrition Inc Recombinant production of steviol glycosides
CA3128532A1 (en) 2011-08-08 2013-02-14 Evolva Sa Recombinant production of steviol glycosides
US10017804B2 (en) 2013-02-11 2018-07-10 Evolva Sa Efficient production of steviol glycosides in recombinant hosts
MY191735A (en) 2014-09-09 2022-07-13 Evolva Sa Production of steviol glycosides in recombinant hosts
EP3232817A4 (en) 2014-12-17 2018-10-10 Cargill, Incorporated Steviol glycoside compounds, compositions for oral ingestion or use, and method for enhancing steviol glycoside solubility
EP4148137A1 (en) 2015-01-30 2023-03-15 Evolva SA Production of steviol glycosides in recombinant hosts
CA2979931A1 (en) 2015-03-16 2016-09-22 Dsm Ip Assets B.V. Udp-glycosyltransferases
BR112017021066B1 (en) * 2015-04-03 2022-02-08 Dsm Ip Assets B.V. STEVIOL GLYCOSIDES, METHOD FOR THE PRODUCTION OF A STEVIOL GLYCOSIDE, COMPOSITION, RELATED USES, FOOD, PET FOOD AND BEVERAGE
WO2017025362A1 (en) 2015-08-07 2017-02-16 Evolva Sa Production of steviol glycosides in recombinant hosts
CA3005993A1 (en) * 2015-11-30 2017-06-08 Cargill, Incorporated Steviol glycoside compositions for oral ingestion or use
WO2017153538A1 (en) * 2016-03-11 2017-09-14 Evolva Sa Production of steviol glycosides in recombinant hosts
US10982249B2 (en) 2016-04-13 2021-04-20 Evolva Sa Production of steviol glycosides in recombinant hosts
CN109312378A (en) 2016-05-16 2019-02-05 埃沃尔瓦公司 Steviol glycoside is generated in the recombination host
MY190181A (en) * 2016-06-17 2022-03-31 Cargill Inc Steviol glycoside compositions for oral ingestion or use
WO2017218888A1 (en) * 2016-06-17 2017-12-21 Cargill, Incorporated Steviol glycoside compositions for oral ingestion or use
WO2018031955A2 (en) 2016-08-12 2018-02-15 Amyris, Inc. Udp-dependent glycosyltransferase for high efficiency production of rebaudiosides
CN109890221B (en) * 2016-10-04 2023-04-14 可口可乐公司 Diterpene glycosides containing an ent-acteosene core, compositions, and methods
CA3039105C (en) 2016-10-14 2024-03-05 Conagen Inc. Biosynthetic production of steviol glycosides and processes therefore
WO2018075874A1 (en) * 2016-10-20 2018-04-26 The Coca-Cola Company Diterpene glycosides isolated from stevia, compositions and methods
AU2016427130B2 (en) 2016-10-21 2022-08-18 Pepsico, Inc. Method for preparing rebaudioside N using enzymatic method
CN110100006A (en) 2016-11-07 2019-08-06 埃沃尔瓦公司 The production of steviol glycoside in recombinant host
WO2018144679A2 (en) 2017-02-03 2018-08-09 Codexis, Inc. Engineered glycosyltransferases and steviol glycoside glucosylation methods
KR20200024789A (en) 2017-06-30 2020-03-09 코나겐 인크. Hydrolysis of Steviol Glycoside by Beta-glucosidase
WO2019055325A2 (en) 2017-09-12 2019-03-21 Biocapital Holdings, Llc Biological devices and methods for using the same to produce steviol glycosides
EP3764815A4 (en) * 2018-03-16 2022-01-26 PureCircle USA Inc. High-purity steviol glycosides
KR20210027270A (en) * 2018-06-08 2021-03-10 퓨어써클 유에스에이 잉크. High purity steviol glycoside
AU2019360932A1 (en) * 2018-10-15 2021-05-06 Amyris, Inc. Stevia rebaudiana kaurenoic acid hydroxylase variants for high efficiency production of rebaudiosides
KR20210125474A (en) * 2018-11-27 2021-10-18 퓨어써클 유에스에이 잉크. High Purity Steviol Glycoside
BR112021011419A2 (en) * 2018-12-12 2021-09-08 Conagen Inc. PRODUCTION METHOD OF R6-2A AND/OR R6-2B, R7-2, R6-1 AND R6-4A AND/OR R6-4B, SYNTHETIC REBAUDIOSIDE AND ORAL CONSUMPTION PRODUCT
KR20210129679A (en) * 2019-02-15 2021-10-28 퓨어써클 유에스에이 잉크. High Purity Steviol Glycoside
CA3131818A1 (en) * 2019-03-29 2020-10-08 Guohong MAO Biosynthetic production of udp-rhamnose
JP2022530986A (en) * 2019-05-03 2022-07-05 アミリス，インコーポレイティド Kaurenic acid 13-hydroxylase (KAH) variant and its use
CN112080480B (en) * 2019-06-14 2023-01-03 中国科学院分子植物科学卓越创新中心 Glycosyltransferase mutants and uses thereof
CN110305855B (en) * 2019-06-26 2022-03-22 昆明理工大学 Gastrodia elata GeCPR gene and application thereof
EP4084629A4 (en) * 2019-12-31 2023-12-27 Air Protein, Inc. High protein food compositions
CN113444131B (en) * 2021-06-24 2022-10-21 鲁东大学 N-acetylglucosamine compounds, and preparation method and application thereof

Family Cites Families (149)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS58149697A (en) 1982-02-27 1983-09-06 Dainippon Ink & Chem Inc Preparation of beta-1,3-glycosyl stevioside
JPS59101408A (en) 1982-12-02 1984-06-12 Junichi Iwamura Plant growth regulator
US5198360A (en) 1990-01-19 1993-03-30 Eli Lilly And Company Dna sequence conferring a plaque inhibition phenotype
US5484956A (en) 1990-01-22 1996-01-16 Dekalb Genetics Corporation Fertile transgenic Zea mays plant comprising heterologous DNA encoding Bacillus thuringiensis endotoxin
US6946587B1 (en) 1990-01-22 2005-09-20 Dekalb Genetics Corporation Method for preparing fertile transgenic corn plants
JPH03277275A (en) 1990-03-28 1991-12-09 Dainippon Ink & Chem Inc Novel enzyme and method for preparing glycoside with same enzyme
US5204253A (en) 1990-05-29 1993-04-20 E. I. Du Pont De Nemours And Company Method and apparatus for introducing biological substances into living cells
US5306862A (en) 1990-10-12 1994-04-26 Amoco Corporation Method and composition for increasing sterol accumulation in higher plants
US5460949A (en) 1990-11-15 1995-10-24 Amoco Corporation Method and composition for increasing the accumulation of squalene and specific sterols in yeast
US5712112A (en) 1992-11-04 1998-01-27 National Science Council Of R.O.C. Gene expression system comprising the promoter region of the alpha-amylase genes
JPH09503905A (en) 1993-07-15 1997-04-22 ネオゼ ファーマシューティカルス Method for synthesizing sugar composition
US7186891B1 (en) 1996-04-12 2007-03-06 University Of Kentucky, Research Foundation Plant cells and plants expressing chimeric isoprenoid synthases
JPH10117776A (en) 1996-10-22 1998-05-12 Japan Tobacco Inc Transformation of indica rice
CA2309791A1 (en) 1997-10-06 1999-04-15 The Centre National De Recherche Scientifique Plant fatty acid hydroxylase genes
US6255557B1 (en) 1998-03-31 2001-07-03 Her Majesty The Queen In Right Of Canada As Represented By The Ministerof Agriculture And Agri-Food Canada Stevia rebaudiana with altered steviol glycoside composition
WO1999053071A1 (en) 1998-04-14 1999-10-21 Kyowa Hakko Kogyo Co., Ltd. Process for producing isoprenoid compounds by using microorganisms and method for detecting compounds having antibacterial or herbicidal activity
TWI250210B (en) 1998-05-06 2006-03-01 Dsm Ip Assets Bv An isolated DNA sequence coding for an enzyme involved in the mevalonate pathway or the pathway from isopentenyl pyrophosphate to farnesyl pyrophosphate
EP0955363A3 (en) 1998-05-06 2004-01-28 F. Hoffmann-La Roche Ag Dna sequences encoding enzymes involved in production of isoprenoids
EP1095002A4 (en) 1998-07-06 2005-08-03 Dcv Inc Method of vitamin production
EP2305825B1 (en) 1998-07-06 2015-01-14 DCV Inc. doing business as Bio-Technical Resourses Method of vitamin production
AR021636A1 (en) 1998-12-17 2002-07-31 Rubicon Forests Holdings Ltd MATERIALS AND METHODS FOR THE MODIFICATION OF CONTENT, COMPOSITION AND METABOLISM OF ISOPRENOIDS
CA2352488A1 (en) 1998-12-23 2000-06-29 The Samuel Roberts Noble Foundation, Inc. Plant transformation process
PT1190068E (en) 1999-04-15 2006-06-30 Calgene Llc SEQUENCES OF NUCLEIC ACIDS FOR PROTEINS INVOLVED IN TOCOFEROL SYNTHESIS
AU4365200A (en) 1999-04-21 2000-11-02 Samuel Roberts Noble Foundation, Inc., The Plant transformation process
DE60036477T2 (en) 1999-08-04 2008-06-12 Bacher, Adelbert, Prof. Dr.med. Dr.rer.nat. ISOPRENOID BIOSYNTHESIS
AU6780700A (en) 1999-08-18 2001-03-13 Paradigm Genetics, Inc. Methods and apparatus for transformation of monocotyledenous plants using agrobacterium in combination with vacuum filtration
WO2001083769A2 (en) 2000-05-03 2001-11-08 The Salk Institute For Biological Studies Crystallization of 4-diphosphocytidyl-2-c-methylerythritol synthesis
DE10027821A1 (en) 2000-06-05 2001-12-06 Adelbert Bacher New intermediate in isoprenoid biosynthesis, useful in screening for potential herbicides, comprises mutant encoding-enzymes sequences for imparting herbicide resistance
US20030033626A1 (en) 2000-07-31 2003-02-13 Hahn Frederick M. Manipulation of genes of the mevalonate and isoprenoid pathways to create novel traits in transgenic organisms
US7109033B2 (en) 2000-08-24 2006-09-19 The Scripps Research Institute Stress-regulated genes of plants, transgenic plants containing same, and methods of use
US6818424B2 (en) 2000-09-01 2004-11-16 E. I. Du Pont De Nemours And Company Production of cyclic terpenoids
US6660507B2 (en) 2000-09-01 2003-12-09 E. I. Du Pont De Nemours And Company Genes involved in isoprenoid compound production
US6689601B2 (en) 2000-09-01 2004-02-10 E. I. Du Pont De Nemours And Company High growth methanotropic bacterial strain
AU2001291114A1 (en) 2000-09-19 2002-04-02 Microbia, Inc. Modulation of secondary metabolite production by zinc binuclear cluster proteins
US20030219798A1 (en) 2000-09-29 2003-11-27 Gokarn Ravi R. Isoprenoid production
US6949362B2 (en) 2000-12-12 2005-09-27 E. I. Du Pont De Nemours And Company Rhodococcus cloning and expression vectors
DE60239386D1 (en) 2001-01-25 2011-04-21 Evolva Ltd CELL LIBRARY
US20040078846A1 (en) 2002-01-25 2004-04-22 Desouza Mervyn L. Carotenoid biosynthesis
US20050003474A1 (en) 2001-01-26 2005-01-06 Desouza Mervyn L. Carotenoid biosynthesis
DE10201458A1 (en) 2001-04-11 2002-10-17 Adelbert Bacher New proteins involved in isoprenoid biosynthesis, useful in screening for inhibitors, also new intermediates, potential therapeutic agents, nucleic acids and antibodies
US7034140B2 (en) 2001-04-24 2006-04-25 E.I. Du Pont De Nemours And Company Genes involved in isoprenoid compound production
CN1630718A (en) 2001-06-06 2005-06-22 Dsmip资产公司 Improved isoprenoid production
WO2003008540A2 (en) 2001-06-22 2003-01-30 Syngenta Participations Ag Abiotic stress responsive polynucleotides and polypeptides
NZ513755A (en) 2001-08-24 2001-09-28 Ann Rachel Holmes Protein expression system in yeast comprising a vector encoding a heterologous membrane protein and its application in screening for drugs
US20040072311A1 (en) 2001-08-28 2004-04-15 Dicosimo Deana J. Production of cyclic terpenoids
US20040010815A1 (en) 2001-09-26 2004-01-15 Lange B. Markus Identification and characterization of plant genes
US7172886B2 (en) 2001-12-06 2007-02-06 The Regents Of The University Of California Biosynthesis of isopentenyl pyrophosphate
ATE444367T1 (en) 2002-08-20 2009-10-15 Suntory Holdings Ltd NEW GLYCOSYLTRANSFERASE GENES
US7351564B2 (en) 2002-09-27 2008-04-01 Dsm Ip Assets B.V. SQS gene
US7098000B2 (en) 2003-06-04 2006-08-29 E. I. Du Pont De Nemoure And Company Method for production of C30-aldehyde carotenoids
WO2004111214A1 (en) 2003-06-12 2004-12-23 Dsm Ip Assets B.V. Feedback-resistant mevalonate kinases
US7569389B2 (en) 2004-09-30 2009-08-04 Ceres, Inc. Nucleotide sequences and polypeptides encoded thereby useful for modifying plant characteristics
EP2365090A1 (en) 2004-05-21 2011-09-14 The Regents of The University of California Method for enhancing production of isoprenoid compounds
WO2006069610A2 (en) 2004-07-02 2006-07-06 Metanomics Gmbh Process for the production of fine chemicals
US20060014264A1 (en) 2004-07-13 2006-01-19 Stowers Institute For Medical Research Cre/lox system with lox sites having an extended spacer region
WO2006016395A1 (en) 2004-08-09 2006-02-16 National University Corporation Tohoku University Udp-glucuronyl transferase and gene encoding the same
WO2006018211A1 (en) 2004-08-19 2006-02-23 Dsm Ip Assets B.V. Production of isoprenoids
US7923552B2 (en) 2004-10-18 2011-04-12 SGF Holdings, LLC High yield method of producing pure rebaudioside A
ATE500321T1 (en) 2004-12-14 2011-03-15 Dsm Ip Assets Bv IMPROVED MEVALONATE KINASE
WO2006092449A2 (en) 2005-03-02 2006-09-08 Metanomics Gmbh Process for the production of fine chemicals
WO2006096392A2 (en) 2005-03-04 2006-09-14 Diversa Corporation Enzymes involved in astaxanthin, carotenoid and isoprenoid biosynthetic pathways, genes encoding them and methods of making and using them
JP4284562B2 (en) 2005-03-04 2009-06-24 独立行政法人産業技術総合研究所 Method for producing UDP-xylose
US7989677B2 (en) 2005-08-17 2011-08-02 Nestec S. A. Nucleic acids and proteins associated with sucrose accumulation in coffee
WO2007024816A2 (en) 2005-08-22 2007-03-01 Co2 Boost Llc A device and process to generate co2 used for indoor crop production and underwater gardening
US8293307B2 (en) 2005-10-11 2012-10-23 Purecircle Sdn Bhd Process for manufacturing a sweetener and use thereof
DK2526778T3 (en) 2005-11-23 2017-03-27 Coca Cola Co Natural High Strength Sweetener Compositions with Improved Time Profile and / or Taste Profile, Methods of Formulation thereof and Applications thereof
KR100784261B1 (en) 2006-01-02 2007-12-11 한국과학기술원 Method for Cell Surface Displaying of Target Proteins Using Bacillus anthracis Exosporium
US7927851B2 (en) 2006-03-21 2011-04-19 Vineland Research And Innovation Centre Compositions having ent-kaurenoic acid 13-hydroxylase activity and methods for producing same
CA2644273A1 (en) 2006-04-05 2008-03-27 Metanomics Gmbh Process for the production of a fine chemical
WO2007136847A2 (en) 2006-05-19 2007-11-29 The Regents Of The University Of California Methods for increasing isoprenoid and isoprenoid precursor production by modulating fatty acid levels
WO2007147275A1 (en) 2006-06-19 2007-12-27 Givaudan Sa Nucleic acid, polypeptide and its use
WO2008008256A2 (en) 2006-07-07 2008-01-17 The Regents Of The University Of California Methods for enhancing production of isoprenoid compounds by host cells
US8257957B2 (en) 2006-09-26 2012-09-04 The Regents Of The University Of California Production of isoprenoids and isoprenoid precursors
US7629156B2 (en) 2006-09-28 2009-12-08 E.I. Du Pont De Nemours And Company Ethanol production in fermentation of mixed sugars containing xylose
US7741119B2 (en) 2006-09-28 2010-06-22 E. I. Du Pont De Nemours And Company Xylitol synthesis mutant of xylose-utilizing zymomonas for ethanol production
CN101200480B (en) 2006-12-15 2011-03-30 成都华高药业有限公司 Rebaudioside A extraction method
JP4915917B2 (en) 2006-12-22 2012-04-11 独立行政法人農業・食品産業技術総合研究機構 Method for producing lacto-N-biose I and galacto-N-biose
EP2124633B1 (en) 2007-01-22 2012-03-07 Cargill, Incorporated Method of producing purified rebaudioside a compositions using solvent/antisolvent crystallization
JP2008237110A (en) 2007-03-27 2008-10-09 Institute Of Physical & Chemical Research Steviol synthetic enzyme gene and method for producing steviol
WO2008144060A2 (en) 2007-05-17 2008-11-27 Tetravitae Bioscience, Inc. Methods and compositions for producing solvents
US20080292775A1 (en) 2007-05-22 2008-11-27 The Coca-Cola Company Delivery Systems for Natural High-Potency Sweetener Compositions, Methods for Their Formulation, and Uses
WO2009005704A1 (en) 2007-07-03 2009-01-08 The Regents Of The University Of California Methods of increasing isoprenoid or isoprenoid precursor production
JP2009034080A (en) 2007-08-03 2009-02-19 Sanei Gen Ffi Inc New glycosyltransferase and method for producing glycoside by utilizing the same
US7964232B2 (en) 2007-09-17 2011-06-21 Pepsico, Inc. Steviol glycoside isomers
EP2193202A2 (en) 2007-09-21 2010-06-09 BASF Plant Science GmbH Plants with increased yield
EP2214681B1 (en) 2007-12-03 2021-10-20 DSM IP Assets B.V. Novel nutraceutical compositions containing steviol and uses thereof
CA2710629C (en) 2007-12-27 2016-06-21 Mcneil Nutritionals, Llc Synergistic sweetening compositions
US20110061124A1 (en) 2008-02-20 2011-03-10 Ceres, Inc Nucleotide sequences and corresponding polypeptides conferring improved nitrogen use efficiency characteristics in plants
TWI475963B (en) 2008-02-25 2015-03-11 Coca Cola Co Rebaudioside a derivative products and methods for making
CA2728285A1 (en) 2008-03-03 2009-09-11 Joule Unlimited, Inc. Engineered co2 fixing microorganisms producing carbon-based products of interest
US9005444B2 (en) 2008-05-13 2015-04-14 Cargill, Incorporated Separation of rebaudioside A from stevia glycosides using chromatography
WO2010021001A2 (en) 2008-08-19 2010-02-25 Kaushik Ramakrishnan S Process for preparing sweetener from stevia rebaudiana
ES2959686T3 (en) 2008-10-03 2024-02-27 Morita Kagaku Kogyo New steviol glycosides
CN101720910B (en) 2008-10-23 2012-07-25 大闽食品（漳州）有限公司 Method for preparing stevioside
US8614085B2 (en) 2009-02-27 2013-12-24 Butamax(Tm) Advanced Biofuels Llc Yeast with increased butanol tolerance involving a multidrug efflux pump gene
US20100297722A1 (en) 2009-05-20 2010-11-25 Board Of Trustees Of Southern Illinois University Transgenic moss producing terpenoids
ES2660698T3 (en) 2009-06-08 2018-03-23 Jennewein Biotechnologie Gmbh HMO synthesis
KR20120027363A (en) 2009-06-16 2012-03-21 이피씨 (베이징) 내추럴 프로덕츠 컴퍼니, 리미티드 Composition comprising rebaudioside d for reducing or eliminating aftertaste and preparation method thereof
SG178376A1 (en) 2009-09-04 2012-03-29 Redpoint Bio Corp Sweetness enhancers including rebaudioside a or d
US20120230922A1 (en) 2009-09-22 2012-09-13 Redpoint Bio Corporation Novel Polymorphs of Rebaudioside C and Methods for Making and Using the Same
RU2596190C9 (en) 2009-10-15 2016-10-20 ПЬЮСЁРКЛ ЭсДиЭн БиЭйчДи Rebaudioside d of high purity and use thereof
US8299224B2 (en) 2009-10-15 2012-10-30 Purecircle Sdn Bhd High-purity Rebaudioside D
US8703224B2 (en) 2009-11-04 2014-04-22 Pepsico, Inc. Method to improve water solubility of Rebaudioside D
EP2504441B1 (en) 2009-11-23 2020-07-22 E. I. du Pont de Nemours and Company Sucrose transporter genes for increasing plant seed lipids
PL2510096T5 (en) 2009-12-10 2018-06-29 Regents Of The University Of Minnesota Tal effector-mediated dna modification
RU2572756C2 (en) 2009-12-28 2016-01-20 Дзе Кока-Кола Компании Sweetness intensifiers, their compositions and application methods
KR101244315B1 (en) 2010-10-19 2013-03-14 이화여자대학교 산학협력단 Ethanol―Resistant Yeast Genes and Uses Thereof
WO2011140329A1 (en) 2010-05-06 2011-11-10 Ceres, Inc. Transgenic plants having increased biomass
EP2571991A1 (en) 2010-05-20 2013-03-27 Evolva SA Method of producing isoprenoid compounds in yeast
WO2011151326A2 (en) 2010-05-31 2011-12-08 Vib Vzw The use of transporters to modulate flavor production by yeast
NZ708078A (en) * 2010-06-02 2017-01-27 Evolva Nutrition Inc Recombinant production of steviol glycosides
US20120021111A1 (en) 2010-07-23 2012-01-26 Aexelon Therapeutics, Inc. Natural Low Caloric Sweetener Compositions for Use in Beverages, Foods and Pharmaceuticals, and Their Methods of Manufacture
US20120083593A1 (en) 2010-10-01 2012-04-05 Shanghai Yongyou Bioscience Inc. Separation and Purification of Stevioside and Rebaudioside A
US9284570B2 (en) 2010-11-30 2016-03-15 Massachusetts Institute Of Technology Microbial production of natural sweeteners, diterpenoid steviol glycosides
KR20130014227A (en) 2011-07-29 2013-02-07 한국생명공학연구원 NOVEL α-GLUCOSYL STEVIOSIDES AND PROCESS FOR PRODUCING THE SAME
CA3128532A1 (en) 2011-08-08 2013-02-14 Evolva Sa Recombinant production of steviol glycosides
CA2843549A1 (en) 2011-08-08 2013-02-14 Shriram RAGHAVAN Methods and materials for recombinant production of saffron compounds
SG11201401927PA (en) 2011-11-23 2014-09-26 Evolva Sa Methods and materials for enzymatic synthesis of mogroside compounds
CN103159808B (en) 2011-12-09 2017-03-29 上海泓博智源医药股份有限公司 A kind of process for preparing natural sweetener
EP3735841A1 (en) 2011-12-19 2020-11-11 PureCircle SDN BHD Methods for purifying steviol glycosides and uses of the same
US20150031868A1 (en) 2012-01-23 2015-01-29 Dsm Ip Assets B.V. Diterpene production
WO2013137487A1 (en) 2012-03-16 2013-09-19 サントリーホールディングス株式会社 Steviol glycosyltransferase and gene encoding same
EP4012041A1 (en) 2012-05-22 2022-06-15 PureCircle SDN BHD High-purity steviol glycosides
CN103710318B (en) 2012-09-29 2017-01-18 中国科学院上海生命科学研究院 Method for producing stevioside compounds by using microorganisms
WO2014086890A1 (en) 2012-12-05 2014-06-12 Evolva Sa Steviol glycoside compositions sensory properties
CA3171770A1 (en) 2013-02-06 2014-08-14 Evolva Sa Methods for improved production of rebaudioside d and rebaudioside m
US10017804B2 (en) * 2013-02-11 2018-07-10 Evolva Sa Efficient production of steviol glycosides in recombinant hosts
CN105247064A (en) 2013-05-31 2016-01-13 帝斯曼知识产权资产管理有限公司 Extracellular diterpene production
US10689681B2 (en) 2013-05-31 2020-06-23 Dsm Ip Assets B.V. Microorganisms for diterpene production
KR101559478B1 (en) 2013-06-24 2015-10-13 한국생명공학연구원 Method for Preparing Natural High Intensity Sweetener Rebaudiside A by Using Enzymatic Conversion
AU2014292150B2 (en) 2013-07-15 2019-04-04 Dsm Ip Assets B.V. Diterpene production
WO2015011209A1 (en) 2013-07-23 2015-01-29 Dsm Ip Assets B.V. Diterpene production in yarrowia
EP3536697A1 (en) 2013-07-31 2019-09-11 DSM IP Assets B.V. Recovery of steviol glycosides
BR112016001950A2 (en) 2013-07-31 2017-08-29 Dsm Ip Assets Bv STEVIOL GLYCOSIDES
WO2015016393A1 (en) 2013-08-02 2015-02-05 サントリーホールディングス株式会社 Method for using hexenol glycosyl transferase
CN103397064B (en) 2013-08-14 2015-04-15 苏州汉酶生物技术有限公司 Method for preparing rebaudioside M through enzyme method
WO2015028324A2 (en) 2013-08-30 2015-03-05 Evolva Sa A method for producing modified resveratrol
WO2015051454A1 (en) 2013-10-07 2015-04-16 Vineland Research And Innovation Centre Compositions and methods for producing steviol and steviol glycosides
US20170067063A1 (en) 2014-03-07 2017-03-09 Evolva Sa Methods for Recombinant Production of Saffron Compounds
BR112017002783A2 (en) 2014-08-11 2017-12-19 Evolva Sa production of steviol glycosides in recombinant hosts
MY191735A (en) 2014-09-09 2022-07-13 Evolva Sa Production of steviol glycosides in recombinant hosts
EP3201212B1 (en) * 2014-10-03 2018-09-19 Conagen Inc. Non-caloric sweeteners and methods for synthesizing
EP3232817A4 (en) * 2014-12-17 2018-10-10 Cargill, Incorporated Steviol glycoside compounds, compositions for oral ingestion or use, and method for enhancing steviol glycoside solubility
EP4148137A1 (en) * 2015-01-30 2023-03-15 Evolva SA Production of steviol glycosides in recombinant hosts
CA2979931A1 (en) 2015-03-16 2016-09-22 Dsm Ip Assets B.V. Udp-glycosyltransferases
BR112017021066B1 (en) * 2015-04-03 2022-02-08 Dsm Ip Assets B.V. STEVIOL GLYCOSIDES, METHOD FOR THE PRODUCTION OF A STEVIOL GLYCOSIDE, COMPOSITION, RELATED USES, FOOD, PET FOOD AND BEVERAGE
CN104845990A (en) 2015-06-11 2015-08-19 山东大学 Application of Arabidopsis glycosyltransferase gene UGT73C7 in improving plant disease resistance
WO2017025362A1 (en) 2015-08-07 2017-02-16 Evolva Sa Production of steviol glycosides in recombinant hosts
CA3005993A1 (en) * 2015-11-30 2017-06-08 Cargill, Incorporated Steviol glycoside compositions for oral ingestion or use

Also Published As

Publication number Publication date
US10364450B2 (en) 2019-07-30
US20170369922A1 (en) 2017-12-28
JP2018503388A (en) 2018-02-08
AU2016211124A1 (en) 2017-07-27
EP3250686A1 (en) 2017-12-06
CN108337892A (en) 2018-07-27
EP4148137A1 (en) 2023-03-15
CN108337892B (en) 2022-06-24
US11807888B2 (en) 2023-11-07
SG11201705606PA (en) 2017-08-30
BR112017016338A2 (en) 2018-03-27
US11041183B2 (en) 2021-06-22
AU2022205176A1 (en) 2022-08-04
US20200017896A1 (en) 2020-01-16
US20230033436A1 (en) 2023-02-02
WO2016120486A1 (en) 2016-08-04
US20240026409A1 (en) 2024-01-25
AU2016211124B2 (en) 2022-04-14

Similar Documents

Publication Publication Date Title
US11807888B2 (en) Production of steviol glycoside in recombinant hosts
US20220195477A1 (en) Production of steviol glycosides in recombinant hosts
US20210155966A1 (en) Production of steviol glycosides in recombinant hosts
US11821015B2 (en) Production of steviol glycosides in recombinant hosts
US20220154234A1 (en) Production of steviol glycosides in recombinant hosts
AU2015314251A1 (en) Production of steviol glycosides in recombinant hosts
US20200291442A1 (en) Production of steviol glycosides in recombinant hosts
US11396669B2 (en) Production of steviol glycosides in recombinant hosts
US20190048356A1 (en) Production of steviol glycosides in recombinant hosts

Legal Events

Date Code Title Description
EEER Examination request

Effective date: 20210118

EEER Examination request

Effective date: 20210118

EEER Examination request

Effective date: 20210118

EEER Examination request

Effective date: 20210118

EEER Examination request

Effective date: 20210118

EEER Examination request

Effective date: 20210118